AU623131B2 - Tyr-peptide analogs - Google Patents

Description

I--

62 FORM 1 FORM S F Ref: 93154 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Address for Service: F Hoffmann-La Roche Co Aktiengesellschaft Grenzacherstrasse 124-184 4002 Basle

SWITZERLAND

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Tyr-Peptide Analogs The following statement is a full description of this invention, including the best method of performing it known to me/us 5845/3 X I 1 ABSTRACT OF DISCLOSURE Analogs of tyrosine sulfate- or tyrosine phosphate residue(s) containing peptides, the novel intermediate compounds used in the preparation of these analogs, as well as a method for suppressing appetite in subjects by administering to the subject an effective amount of CCK analog wherein one or more of any tyrosine sulfate residue(s) present is/are replaced by a residue of the invention.

r 0£ *t 4 4O 00 *D 4 0 0 00 4 0 4 4 0 0000 00 a6 0 04 0 0

K

I_

RAN 4105/116 1A Peptides are ubiquitous biological molecules and have, in recent years, become the subject of extensive research and investigation. For example, the possibilities for utilizing natural biological substances, such as peptides, as therapeutics for various diseases is being aggressively explored.

Elucidation of the amino acid sequences of such peptides such as Growth Hormone, Growth Hormone Releasing Factor, or Cholecystokinin (CCK) has lead to advancements in the understanding of how these molecules work in treating various disorders. However, peptides suffer from rapid 15 degradation upon exposure to the internal milieu often 0 0 resulting in low bioavailability. It has been recently o° discovered that in many instances if the amino acid constituency of many naturally occurring peptides is altered 0 0 o. o by single or multiple amino acid replacements at different o 0 0 sites, the analogs of the natural peptide may degrade less rapidly and hence exhibit greater bioavailability and efficacy.

S00 0 0 For example, CCK is a family of peptide hormones which vary 25 in length up to 58 amino acids. The amino acid sequence of CCK first discovered had a length of 33 amino acids. CCK as well as fragments thereof, such as CCK-8 and CCK-7, have been shown to have satiety-inducing effects when 0000 0o o administered peripherally to animals. CCK-8 has the amino 0 00 acid sequence: 26 27 28 29 30 31 32 33 Asp-Tyr-(SO 3 H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 AB/10.4.1989 i -I 4 CCK-7 corresponds to the above.

2amino acid sequence 27-33 shown I I 1 44 4 4 440 4 04 4 4 4 o 4 0 o 44 4 0 1 4 44 4044 4044 4 $04 00 0 0 4 4 4 40 While CCK analogs are known to have satiety inducing effects, they exhibit low bioavailability and are poorly absorbed. Tyrosine sulfate containing peptides are well known to suffer loss of the sulfate moiety upon storage, particularly when stored in solution. This has led to the synthesis of various CCK analogs wherein the attempt to improve properties such as stability and bioavailability has been made. A multitude of CCK analogs with various amino acid replacements resulted in compounds with altered properties which enhance their potential usefulness in human therapeutics.

The instant invention comprises analogs of tyrosine sulfate- or tyrosine phosphate residue(s) containing peptides. Examples of tyrosine sulfate residue(s) containing peptides include peptides such as gastrin, cholecystokinin. platelet factor 4, or hirudin. Many tyrosine phosphate residue(s) containing peptides are also known such as human insulin receptor, progesterone receptor, or Lipocortin-1. All of these peptides may yield analogs according to the instant invention.

For example, a tyrosine sulfate residue occupies position 27 of the amino acid sequence of CCK or the corresponding position in its shorter analogs such as CCK-7 and CCK-8. When this tyrosine sulfate residue is replaced by a residue of the invention, the result is a CCK analog which may exhibit greater bioavailability, and hence may lend itself more readily to absorbtion following oral administration.

The instant invention comprises analogs of tyrosine sulfate- or tyrosine phosphate residue(s) containing peptides wherein one or more of such residue(s) is/are replaced by residue(s) of the formula: r^..rre~iskT~~Vlcn~n~w(r 3

A

Y

Z

0,44 e~ aff 0 0 0 0 0 0 0 0 o o h 14 t wherein A, B, Z, and Y are as described hereinafter, and pharmaceutically acceptable salts thereof.

The instant invention also comprises the novel intermediate compounds.

The instant invention also comprises a method of suppressing appetite in subjects by administering an appetite suppressing effective amount of a CCK analog wherein tyrosine Ifate residue(s) is are replaced by residue(s) of the invention.

The following symbols and terminology as utilized in this specification are defined as follows: 1. cyclic peptide 4 4r 444 means a peptide where the omega carboxy terminus of one amino di-acid in the peptide chain is attached to the omega amino terminus of another di-amino acid in the peptide chain via the formation of an amide bond. The bonding between the two amino acids in the chain illustrated by the designation ILi 4 2. lower alkyl 3. aryl 4. lower alkoxy f So o 1 t I i l «tl d results in a ring structure.

means straight or branched chain saturated hydrocarbon residues containing from 1 to 7 carbon atoms.

means substituted or unsubstituted phenyl or napthyl wherein the substituents are one or more halogen, lower alkyl, lower alkoxy or nitro.

means a lower alkyl oxy group such as methoxy, ethoxy, propoxy, etc.

means a straight or branched chain unsaturated hydrocarbon residue containing from 2 to 7 carbon atoms.

means the 3 position of the phenyl on phenylalanine is substituted with COOH.

means the 4 position on the phenyl of phenylalanine is substituted with COOH.

means the 4 position on the phenyl of phenylalanine is substituted with CH 2

COOH.

lower alkenyl 6. Phe(3-COOH) at4 4 5 0 Y, 7. Phe(4-COOH) 8. Phe(4-CH 2

COOH)

L i_ 9. Phe(4-CH 2 COOC 2

H

5 Phe(4-CR CHR COOH) 11. Phe(4-CF 2

COOH)

means the 4 position on the phenyl of phenylalanine is substituted with CHR COOC 2 H means the 4 position on the phenyl of phenylalanine is substituted with CH 2 CH 2

COOR.

means the 4 position on the phenyl of phenylalanine is sub~itituted with CF 2

COOH.

means the 4 position on the phenyl of phenylalanine is substituted with a group.

means that 4 position on the phenyl of phenylalanine is substituted with a CR 2-[tetrazolyl] group.

4 4 4 44 000 0 4 0 0 0 4 4 00 40 o 0 4 15 12. Phe(4-tetrazole) 13. Phe(4-CH 2-tetrazole) 14. peptide means a linear or cyclic peptide.

Ac 1. Acmeans acetyl.

16. or (L) 0 t 4(1 1 a 4 00 0 0 00 00 0 4 0 4 0800 o t

O

*9 t ror

(I

6 f

I

6 preceding the amino acid designation means that this amino acid exists in that specific isomeric form. i.e.

(D,L)Phe means that the amino acid phenylalanine exists as a racemic mixture; (D)Phe means that the amino acid Phenylalanine exists as the R stereoisomer; (L)Phe means that the amino acid Phe exists as the L stereoisomer or implied S configuration. In case that there is no designation preceeding the amino acid the L isomer is implied (S configuration). All amino acids are represented by their commonly understood three letter designations.

where this term is used it includes enantiomers and racemates of the compounds.

means the amino acid tyrosine wherein a sulfate ester is present on the 4-position of the aromatic ring.

means the amino acid tyrosine wherein a phosphate ester is present on the 4-position of the aromatic ring.

17. Compounds 18. Tyrosine Sulfate 19. Tyrosine Phosphate nryin~mr~i~unna^-a*I-~ rp rrrrsr~ ~IC--n~iis~:~xrii Desamino means an amino acid which lacks the alpha-amino functional group. For example, in the context of the instant invention "Desamino Phe" means that the phenylalanine lacks the alpha amino functional group.

means a tetrazole residue with a functional group R attached to a nitrogen in position 1 or 2.

N

21. N

N

R2 S* I 00 00 o a o a The instant invention comprises analogs of tyrosine sulfate- or tyrosine phosphate residue(s) containing peptides wherein one or more of such residue(s) is/are replaced by residue(s) of the formula

Y

z wherein one of A or B is selected from the group consisting of 2

-CO

2

R

1 or

N

-IC(Ro)2]r-<I'

N

R2 and the other of A or B is H; I. 8 Z is H, -NH 2 lower alkyl, -NHCOR 3

-NHCO

2

R

4 or 0

-N

R9

O

LI 0

II

R

2 is

R

3 is H or F; H, substituted or unsubstituted lower alkyl with the substituents selected from the group consisting of hydroxyl, halogen, or aryl; H, lower alkenyl, lower alkyl or lower alkyl substituted by 1 to 3 aryl groups; H, lower alkyl, alkyl substituted by one or two aryl groups or aryl; lower alkyl or alkyl substituted by one or two aryl groups; each independently H, lower alkyl, or taken together and including the carbon ?toms to which they are attached may form a six membered ring which may be aromatic; and r are 0, 1 or 2; -COOH, alkoxycarbonyl, -CONH 2 or -CO-; proviso that if Z is other than -NH- then Y is or if Y is s and Y is with the other than -CO- then Z is -NH-; and pharmaceutically acceptable salts thereof.

According to a second embodiment of this invention, there is provided a compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide according to the first embodiment, said compound having the formula: wherein one of A' or, is selected from -[C(Ro) 2

-CO

2

R

1 or

NN

LMM/597Z

N

JC(Ra)2r1

R

2 1 -i 8A and the other of A' or B' is H wherein R 0 is H or F, R 1 is H, substituted or unsubstituted lower alkyl with the substituents selected from hydroxyl, halogen, or aryl, R 2 is H, lower alkenyl, lower alkyl or lower alkyl substituted by 1 to 3 aryl groups, r is 0-2 s is 1-2 R' is H, lower aikyl or lower alkyl substituted by 1 to 3 aryl groups, Z' is H, lower alkyl, -NH 2

-NHCOR

3

-NHCO

2

R

4 or 0 S R 0

N

o o R9 e 0 00 .o 10 and R 3 and R 4 are H, lower alkyl or lower alkyl substituted by one, two or three aryl groups; and R 5 and R9 are each independently H, lower alkyl, or taken together and including the carbon atoms to which they are attached may form a six membered ring which may be aromatic.

According to a third embodiment of this invention, there is 15 provided a compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide according to the first embodiment, said compound having the formula: j C C RI 'A 3 Z' OH 0 wherein Z' and R 1 are as defined in the second embodiment.

According to a fourth embodiment of this invention, there is provided a compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide according to the first embodiment, said compound having the formula:

CO

S0-R' 0 LMM/597Z 7 _1 8B wherein R' and Z' are as defined in the second embodiment.

According to a fifth embodiment of this invention, there is provided a compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide according to the first embodiment, said compound having the formula:

A'

is H or lower alkyl.

e o c a wherein and R' are as defined in the second embodiment and R 8 is H or lower alkyl.

According to a sixth embodiment of this invention, there is 10 provided a compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide according to the first embodiment, said compound having the formula: wherein and R' are as defined in the second embodiment and R 8 According to a seventh embodiment of this invention, there is provided a process for the manufacture of a compound according to the first embodiment, which process comprises treating the resin bound peptide with appropriate cleavage reagent(s) and if desired, converting such a peptide obtained into a pharmaceutically acceptable salt.

LMM/597Z 8C According to a eighth embodiment of this invention, there is provided a pharmaceutical composition containing a compound according to the first embodiment and a non-toxic, inert, therapeutically acceptable carrier material.

According to a ninth embodiment of this invention, there is provided a pharmaceutical composition for suppressing appetite containing a compound according to the first embodiment and a therapeutically inert carrier material.

According to a tenth embodiment of this invention, there is provided the use of a compound according to the first embodiment as a therapeutically active substance.

According to an eleventh embodiment of this invention, there is provided the use of a compound according to the first embodiment in the S. suppression of appetite.

According to a twelfth embodiment of this invention, there is o° provided a method for suppressing appetite in subjects by administering an appetite suppressing effective amount of an analog of CCK wherein the tyrosine sulfate residue is replaced by a residue of Formula I as defined in the first embodiment.

20 A preferred embodiment of this invention is analogs of tyrosine 4 sulfate residue(s) containing peptides wherein one or more tyrosine sulfate residue(s) are/is replaced by residue(s) of formula I.

1 ,A further preferred embodiment of the present invention is analogs S' of CCK wherein the tyrosine sulfate residue(s) is/are replaced by (a) residue(s) of formula I wherein Z is H, NHCOR 3 or NHCO 2

R

4 and R 3 is lower alkyl, R 4 is lower alkyl optionally substituted with one to three aryl groups and such analogs wherein R 3 is CH 3 34 3 LMM/597Z i Ca~i3 as~aRa~ r~nrraasi-~x i 9 Another preferred embodiment of the present invention are analogs of CCK wherein the tyrosine sulfate residue(s) is/are replaced by a residue of formula I, wherein Z in formula I is H or NHCOR 3 with R 3 lower alkyl or H.

Further preferred are analogs wherein one of A or B in formula I is 0)2s-CO R1 and the other of A or B is H.

Further preferred are analogs wherein A in formula I is

-[C(RO)

2

CO

2

R

1 and B in formula I is H.

Also preferred are analogs wherein s in formula I is 0 and R 1 is H.

More preferred are analogs of CCK wherein tyrosine So' sulfate residue(s) is/are replaced by residue(s) of formula I wherein s is 1, R is H or F, and R is H.

0 ~0 o0 o Most preferred are analogs wherein R in formula I is H, yielding the preferred analogs of the formula: o 0 Ac-(D)Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 25 Ac-Phe(4-CH COOH)-Met-Gly-Trp-Met-Asp-Phe-NH Ac-(D,L)Phe(4-CH 2 COOH)-Lys-Gly-Trp-Met-Asp-N-methyl- 2 Desamino Phe(4-CH2COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Desamino Phe(4-CH COOH)-Nle-Gly-Trp-Nle-Asp-N-methyl -Phe-NH 2 Also preferred are analogs wherein s in formula I is 1,

R

0 in formula I is H, and R 1 in formula I is -CH 2

CH

3 yielding preferred analogs of the formula: i=lr;;DcxUr;)=inne8srr-~4Fa~-aar"------- rr 10 Ac-(D)Phe(4-CH2COOC2H 5 )-Met-Gly-Trp-Met-Asp (COOC

H

5 )-Phe-NH 2 Ac-Phe(4-CH 2

COOC

2

H

5 )-Met-Gly-Trp-Met-Asp-

(COOC

2

H

5 )-Phe-NH 2 Also preferred are analogs wherein B in formula I is CO2R 1 and A in Formula I is H.

More preferred are analogs wherein s in formula I is 1 and R 1 in Formula I is H: Ac-(D,L)Phe(3-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 15 Also preferred are analogs wherein s in formula I is 2 o and R is H and R 1 is H.

00 08 0 o Also preferred are analogs of CCK wherein the tyrosine 0 sulfate residue(s) is/are replaced by residue(s) of formula I wherein one of A or B in Formula I is N

N

Nt

N

R2 and the other of A or B in Formula I is H.

*a0° Further preferred are analogs wherein A in formula I is 0 30 N-N -lC(Ro)2}r N N

R

2 and B in formula I is H.

Most preferred are analogs wherein r in formula I is 0 and R 2 in formula I is H yielding the preferred analogs of the formula: i Ii a 6466 6 6 o 46 off 6 06* 6 00 ~0 0 6 0 6 0 0 0CC 64 o CC 6 0 6 11 Ac-Phe(4-tetrazole) -Met-Gly-Trp-Met-Asp-Phe-NH 2 Desamino-Phe (4-tetrazole) -Met-Gly-Trp-Met-Asp-Phe-NH2 Desamino-Phe(4-tetrazole)-Lys-Gly-Trp--Met-Asp-N-methyl- Phe-NH 2 1 Ac-(DL)Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N-methyl- Phe-NH 2 Also preferred are analogs containing the residue of formula I wherein r in formula I is 1 and R2in formula I is H and Ro is H yielding preferred analogs of the formula: 15 Ac- (D)Phe (4-CH 2 tetrazole) -Met-Gly--Trp-Met-Asp- Phe-NH 2 Ac-Phe(4-CH 2 tetrazole)-Met--Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D)Phe(4-CH 2-tetrazole)-Nle--Gly--Trp-Nle-Asp-N-methyl- P he -NH 2 Ac-Phe(4-CI- 2 tetrazole)-Nle-Gly-Trp-Nle-Asp-N-methyl-Phe -NH 2 Desamino-Phe(4-CH 2- tetrazole)-Nle-Gly-Trp-Nle-Asp-Nmethyl-Phe-NIH 2 The residues incorporated into the analogs of tyrosine sulfate- or tyrosine phosphate residue(s) containing peptides according to the invention may be synthesized as follows:

A

~C llll- rrp~~( li~.l~i i.

12 Scheme I BrCHj

R

6 N OR, Aryl V 0 *tr .4 1 0 a Go Qo 01 00 00 00 o 0 01 0 4 *0 4 00 Aryl

CO

2

F'

C d

H

2

N-

,COR8 C02S Vi' VIll 4 010 000 00 0 0e 1 a 00 'C0 2

H

I

-13- In Reaction Scheme I A' and B' are identical with A and B in formula I with the proviso that R and R are not H.

R' may be a substituted or unsubstituted lower alkyl with the substituents selected from the group consisting of hydroxyl, halogen, or aryl: or any suitable protecting group chosen so as to be easily removed selectively without affecting R 1

R

2 or Z' in step

R

6 may be H or aryl, and Z' is-NH 2

-NHCOR

3 -NHCO2R 4 or 0 1

-R

-RN

0 In Reaction Scheme I the formula III compound is reacted in step with a halogenating agent dissolved in a nonpolar inert organic solvent which contains a catalytic 0 9 o quantity of a radical initiator at a temperature ranging from 60 0 C to 80 0 C until most of the formula III compound is consumed. A suitable halogenating agent is N-bromoo'I. succinimide, a suitable nonpolar inert organic solvent is carbon tetrachloride, and a suitable catalytic radical initiator is azobisisobutyronitrile or benzoyl peroxide.

The resulting formula IV compound can then be isolated by conventional methods such as chromatography or distillation.

In step the formula IV compound is reacted at about room temperature with a formula V compound wherein R 6 is aryl, in a biphasic mixture of, for example, methylene chloride and water which contains an inorganic base, such as alkali metal hydroxide and a phase transfer catalyst, such as a tetraalkylammonium salt [see the procedure described in O'Donnell, et al., Tetrahedron Letters, 2641 (1978)]. The resulting formula VI compound can be isolated by conventional methods, such as chromatography or can be used directly in step -14- Alternatively, the formula V compound wherein R 6 is H, or aryl, can be deprotonated below room temperature with a suitable strong base, such as lithium diisopropylamide or potassium hexamethyldisilazide, in an inert ethereal solvent, such as diethylether, tetrahydrofuran or dimethoxyethane to which is added a metal coordinating agent such as hexamethylphosphoric triamide. A solution of a formula IV compound in an ethereal solvent such as diethyl ether, tetrahydrofuran or dimethoxyethane, can then be added to the mixture preferably at a reaction temperature of about -780 C. After complete reaction and aqueous workup, the resulting formula VI compound may be isolated by conventional methods or may be used directly in step In step the compound of formula VI is treated with at least one equivalent of a strong acid dissolved in a suitable organic solvent which contains excess water.

o Suitable acids are toluenesulfonic acid or hydrochloric o acid, and tetrahydrofuran, ether or acetonitrile are 4 20 suitable solvents. After complete reaction, the resultant compound of formula VII can be isolated as its salt by conventional methods such recrystallization or may be used 0 0 ,4 directly in step wherein the formula VII compound is p o o treated with an acylating agent, such as acetic anhydride or 25 t-butylpyrocarbonate, in the presence of a base which may be a tertiary amine such as triethyl amine; at a temperature ranging from 0 C to room temperature. The choice of solvent Sis not critical for this reaction and is normally based on considerations of solubility and reactivity of the reaction components. Dichloromethane is useful for laboratory scale preparations. Isolation of the resulting formula VIII compound can be accomplished using conventional methods.

L 15 In step the conversion of the formula VIII compound to a formula IX compound is carried out by selective ester hydrolysis. Appropriate conditions for this hydrolysis depend on the particular choice of R R 2 and Z' in the compound of formula VIII. Optimally, these groups are chosen so as to facilitate the selective conversion of R' to hydrogen without affecting R 1

R

2 or Z using methods known in the art. For example, where R' is a mono- or diarylmethyl group it can be removed through treatment with hydrogen gas at atmospheric pressure in a polar organic solvent such as ethanol in the presence of of a noble metal catalyst such as 10% Pd/C. These reaction conditions may be controlled so as not to affect R 1 or R when these groups are lower alkyl, mono-, di-, or trihaloalkyl, or S 15 trialkylsilylalkyl and will not affect Z' when R 3 and R are appropriately selected, as for example lower alkyl. The I resulting formula IX compound can be isolated by conventional methods.

Alternatively, when R' is a small alkyl group such as methyl and R 1 is a bulky alkyl group such as a tertiary S4" butyl group; selective base hydrolysis may be effected by I treatment of the formula VIII compound with an alkali metal j hydroxide, in the presence of a polar solvent until 1 25 hydrolysis of R' is complete. When done carefully these reaction conditions will not affect R 1

R

2 or Z' f Isolation of the formula IX compound can then be ;accomplished using conventional methods.

i 16 Scheme II ,.,a4 ~~(CH)fCN Br x g 0 000 0 cs 0~' 0 600 0 0.0 0~.

0 0'

OC

o o .4 C' 00 02 o OC 00 0 00 ~4 V Q~ o o 0 0' Br

N,

IK 2 x~i h j (CH?2)r

I

H0 2 CN R2 xili HO (CH2)1-K 1 N" A xlv i 4 N x v 0040

'C.

00 17 In Reaction Scheme II, r and R'2 are as defined in formula I for r and R 2 respectively except that R' 2 should not be H, and the side chain may be attached to either position 3 or 4 on the aromatic ring as indicated.

In step the formula X compound is treated with sodium azide and ammonium chloride in a polar aprotic solvent such as dimethylformamide at a temperature of about 90-100 0

C.

The resultant formula XI compound can then be isolated by conventional techniques. In step the tetrazole of formula XI is treated with an excess of an alcohol capable of forming a stabilized carbonium ion in the presence of a strong acid (such as trifluoroacetic acid), at about room temperature. The major product from this process will be a formula XII compound, however, the regioisomer resulting S 15 from alkylation at N-i of the tetrazole will also be formed in varying amounts depending on the selection of r and the :particular reaction conditions. Formula XII compounds and a C their regioisomers may be separated but this is generally G oo not necessary as both isomers will yield the same product when R'2 is converted to hydrogen. Alternatively, a primary or secondary alcohol can be reacted with a formula o, XI compound in the presence of a dialkyl azodicarboxylate So and a phosphine in an inert solvent according to the procedure described by O. Mitsunobu, Synthesis, (1981) 1; to 00" 25 give a formula XII compound, wherein R 2 is derived from a primary or secondary alcohol.

primary or secondary alcohol.

c 2 S 9t In step the formula XII compound is carbonylated according to the method of Schoenberg, et al. J. Org. Chem., 39, 3318 (1974). Thus a compound of formula XII is maintained under an atmosphere of carbon monoxide ranging from 100-200 psi in the presence of a tertiary amine base in a polar solvent system that contains a trialkyl or triaryl phosphine and a source of catalytic palladium zero. This mixture is maintained at about 1000 C from 1 to 3 days. The resulting formula XIII compound may be isolated by conventional techniques.

r~ ~~-ira 18 In step the formula XIII compound is treated with a metal hydride reducing agent in an inert solvent at room temperature. The resulting compound of formula XIV compound may be isolated by conventional techniques. In step the formula XIV alcohol is treated with triphenylphosphine and carbon tetrabromide in an inert solvent at or below room temperature, preferably at about O°C. The resulting formula XV compound can be isolated by conventional means.

011 .0 a 0 0 0 A 0 -D C 0 A 0 a Ar 0 0 4J A a 0 AUs A6 A 0 AA L 19 Scheme III 4 3

O-R'

0 xvi 0^C0 2 R I

XVII

00 0 o 00 o0 0CC 0 0 0 a' 0 0 N~3 0 0-R' 0 44 00 2

R

1 N 3 0

VI

C0 2 R 1 4

N

0 3 Z, irOH 0

XIX

4 C0 2 R 1 N0 3 Z' O-R' 0~lI 0 Ri 1 0H

XXII

XXI

0000 0000 00 0 0 0 0 0 *0 4 C0 2

R

1

'N

0 3

OH

z 0

XXIV

k~ 20 In Reaction Scheme III, R' is as previously defined, Z'

O

is H, lower alkyl, -NHCOR 3 -NHCO2R or 3' 2 4' -N

-R

-R9 9 0 and R is bromide, iodide, or a perfluoroalkylsulfonate ester which may be attached to the phenyl at either position 3 or 4 as set forth in formula XVI, and R1 is the same as

R

1 in formula I with the proviso that R'1 is other than H. In step the formula XVI compound is reacted with an acrylate of formula XVII in the presence of a source of palladium zero and a tertiary amine base. When R is a I 7 perfluoroalkylsulfonate ester, this procedure is carried out in a polar aprotic solvent at a reaction temperature of 70-100 0 C as described by Chen and Yang, Tetrahedron Letters, 27, 1171 (1986). When R is bromide or iodide, the S, reaction can be carried out as described by Heck, Organic Reactions, 27, (1982) 345. The side chain of the resulting 0o a0 o formula XVIII compound becomes attached to the site of the S 20 departing R7. The formula XVIII compounds may be isolated O°o by conventional means.

Step can be carried out in one or two steps *oo 1 depending on the particular choice of R and R 1 25 When R is mono- or diarylmethyl and R 1 is lower alkyl, halosubstituted alkyl, or hydroxy substituted alkyl, and Z does not contain a mono-, di- or triarylmethoxy group, catalytic hydrogenation over a noble metal catalyst, in a polar solvent at a hydrogen pressure of from one to ,i 30 three atmospheres results in the simultaneous reduction of the double bond and hydrogenolysis of the R group yielding a formula XIX compound after a suitable isolation procedure. Alternatively, when R is methyl or ethyl and

R

1 is a bulky substituent such as, tertiary butyl, catalytic hydrogenolysis may be carried out as above, and a 7 i .i I-o~ ararPaarr r 21 separate selective hydrolysis step may be carried out as described above for step in Scheme I to yield the formula XIX compound.

In step the formula XVI compound where R 7 is perfluoroalkylsulfonate ester is treated with an allyl trialkylstannane, a source of palladium zero, and an excess of lithium chloride in a polar aprotic solvent at about 0 C. The resulting formula XX compound can be isolated by conventional techniques. In step the formula XX compound is reacted with a suitable oxidizing agent and a catalytic amount of ruthenium trichloride in a two phase system as described by Carlsen et al., J. Organic Chemistry, 46, (1981), 3936. Depending on the particular choice of reaction conditions this process may lead either directly to a compound of formula XXI or to an intermediate aldehyde So which may be oxidized to a formula XXI compound according to the procedure described by Bal et al., Tetrahedron, 37, 2091 o. (1981). The resulting formula XXI compounds can be isolated by conventional techniques.

0 on In step an acid of formula XXI is converted to an ester of formula XXIII. The choice of R should be such that it will survive the reaction conditions required So 25 to generate an acid of formula XXIV and will thus depend on the selection of R in the formula XXI compound. The ,,formula XXI compound can be reacted with an alcohol of formula XXII in the presence of a coupling reagent and a catalyst in an inert solvent. Alternatively the formula XXI compound may be reacted with a dimethylformamide Sdialkoxyacetal in a inert solvent at a reaction temperature ranging from 60 to 80 0 C. The resulting formula XXIII compound can be isolated by convention means.

In step the formula XXIII compound is converted to the formula XXIV compound as described in step of scheme

I.

22 Schieme IV 0 04 o oo o 0 0 CoO o 40 00 0 0 0 0 00 00 o 0 0 00 0'o 0 0 o 0 0 o a 0 0404 0000 0000 0000 00 0 O 0 0

A'

R

7 xxv

A'

xxvii' Re~ Xxvi s

CO,

2

R'

Re xxviI C0 2

H

xxix I"-U Cn~- -1 _I; 23 In reaction scheme IV, R and R' are as previously defined and R 8 is hydrogen or lower alkyl.

The compound of formula XXV can be prepared by known methods. In Step q the compound of formula XXV is treated with a compound of formula XXVI under the general conditions described for step k in scheme III to give a compound of formula XXVII which can be isolated by conventional means.

In step formula XXVII compound is reduced to a formula XXVIII compound. When A' and B' contain groups resistant to catalytic hydrogenation, this process is conveniently carried out by hydrogenation over a noble metal catalyst (for example palladium/carbon) in a suitable solvent until reduction is complete. The resulting compound of formula XXVIII can be isolated by conventional techniques such as recrystallization. When R' is susceptible to hydrogenolysis, 0° the conditions of step may lead directly to a compound of formula XXIX. Otherwise, a selective hydrolysis may be a o S carried out using the conditions described for step in o 20 reaction scheme I to give a compound of formula XXIX.

0 4# 0 C The instant invention also comprises all the novel intermediate compounds which are used to prepare the compounds of the invention.

These novel intermediates are compounds represented by ,formulas VII, VIII, and IX in scheme I and formulas XIX, XXI, XXIII, and XXIV in scheme III and formula XXIX in scheme IV wherein the substituents are as defined in 30 schemes I-IV except as noted.

Further novel intermediates are the following compounds: L

-I

24 a compound of the formula: wherein one of A or, B is selected from the group consisting of -[C(R0)2]sCO2R' 1 or N

N

N

R

2 V e a 9000 eo a 0 a 0 0 00 0 o a e a 0 a9 Q a 4 q a 0 00 O a 0a A 9 O 0 4 4 a and the other of A' or B' is H wherein R O is H or F, 20 R'1 is H, substituted or unsubstituted lower alkyl with the substituents selected from the group consisting of hydroxyl, halogen, or aryl, R 2 is H, lower alkenyl, lower alkyl or lower alkyl substituted by 1 to 3 aryl groups, r is 0-2 s is 1-2 (c 0 0 R' is H, lower alkyl or lower alkyl substituted by 1 to 30 3 aryl groups.

Z' is H, lower alkyl, -NH 2

-NHCOR

3 -NHCO R 4 0

N

-R

9

O

-L

C-C

i. 25 and R 3 and R 4 are H. substituted or unsubstituted lower alkyl with the substituents selected from one, two, or three aryl groups; and R 5 is each independently H, lower alkyl. or taken together may form a six membered ring which may be aromatic, or a compound of the formula 4 c 0 2R' 3

OH

0 wherein Z' and R' are as defined under or a compound of the formula 6 i 20 i d C0 2

H

o S Z' O-R' 0 wherein R and Z are as defined under or a compound as defined under wherein one of A' or B' Sis N9 -[C(Ro)2r- 30 N N 4 R 2 and the other of A' or B' is H.

wherein Z R2, and R are as defined under or IIL C1~1 I 26 a compound of the formula: wherein and R' are as defined under and R is H or lower alkyl, or a compound as defined under wherein A' is 0q00 4 0 so a

I

4 04 '41 11 I -[C(Ro) 2 ]r R2 and 20 or B' is H, and RO, r, and R 2 are as defined under a compound as defined under wherein r=0, or a compound as defined 25 and R 8 is H said compound under wherein R' is t-butyl having the formula: or i 27 a compound of the formula: wherein and R' are as defined under and R 8 is H or lower alkyl, or a compound as defined under wherein A' is 004q 0 004 a 1q 00 0 a 0 0 0° 0 o00 0 0 0 o 0 00 1 04 1 1 1 1! 0 4 0- 4 4 444(

N

dC(Ro) 2 1r~ N 20 and B' is H and Ro, r, and R 2 are as defined under or a compound as defined under wherein r=0, or 25 a compound as defined under wherein R' is lower alkyl and R 2 is t-butyl said compound having the formula:

,C(CH

3 3

N-N

I1 N. oN Sor a compound as defined under wherein R' is H and R 2 is t-butyl said compound having the formula: -28

C(CH

3 )3

N-N

N ,N C0 2

H

*or a compound as defined under wherein r=l and Ris H, or 0.0 a compound as defined under wherein R' is lower alkyl and R is t-butyl said compound having the formula: 00 0 0 0 NzN 0 0 e N3 00 0 0 0- 0 25 *or 0*0(q) a compound as defined under p wherein RI is H and R 2 is t-butyl said compound having the formula:

N

0*0N N C(CH 3 )3

NI

01 4

CC,_H

29 The residues of formula I may be incorporated into peptides in a protected form, that is R or R may be substituted alkyl such as tertiary butyl and Z may be hydrogen, NHCR 3 or NHCOR as appropriate. Such a residue is then incorporated into the growing peptide chain according to methods known in the art. The protecting groups are then removed according to suitable known methods.

The instant invention also comprises a method for suppressing appetite in subjects by administering to the subject an appetite suppressing effective amount of an analog of CCK wherein the tyrosine sulfate residue(s) is/are replaced by residue(s) of formula I or of an 0r pharmaceutically acceptable salt thereof.

0 Particularly preferred is a method for suppressing Sappetite in subjects by administering to a subject an 20 appetite suppressing effective amount of the following CCK analogs wherein the tyrosine sulfate residue has been replaced by a residue of formula I Ac-(D)Phe-(4-CH2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 .00, 25 Ac-Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 2 Ac-(D)Phe(4-CH2COOC H )-Met-Gly-Trp-Met-Asp 2 SAc-Phe(4-CH COOC 2 H5)-Met-Gly-Trp-Met-Asp 2 2 2 Ac-(D,L)Phe(4-CF2COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D)Phe(4-tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH Ac-Phe(4-tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D,L)Phe(4-CH COOH)Lys-Gly-Trp-Met-Asp-N-methyl-Phe- 2 a- NH2 Ac-(D,L)Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N-methyl-Phe-

NH

2 Ac(D,L)Phe(3-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH L 30 Desamino Phe(4-CH 2 COOH)-Nle-Gly-Trp-Nle-Asp-N-methyl- Phe-NH 2 Desamino Phe(4-tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Desamino Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N-methyl- Phe-NH 2 Ac-(D)Phe(4-CH tetrazole)--Met-Gly-Trp-Met--Asp-Phe-NH 2 Ac-Phe(4-CH tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D)Phe(4-CH 2 tetrazole)-Nle-Gly-Trp-Nle-Asp-N-methyl- Phe-NH 2 Ac-Phe(4-CH 2 -tetrazole)--Nle-Gly-Trp-Nle-Asp-N-methyl- Phe-NHI 2 Desamiino-Phe(4-CH tetrazole)-Nle-Gly-Trp-Nle-Asp-Nmethyl-Phe-NH 2 or of their pharmaceutically acceptable salts.

Most preferred is a method for suppressing appetite in 2 subjects by administering to the subject an appetite suppressing effective amount of the following CCK analogs wherein the tyrosine sulfate residue has been replaced by a residue of formula I Ac-(D)Phe(4-CH 2 COOH)--Met-Gly-Trp-Met-Asp-Phe-NH 2 2, 2 Ac-Phe(4-CH COO H--Met-Gly-Trp-Met-AsphNH (COOC H )Phe-NH 2 25 2 Ac-Phe(4-tetrazole )-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D,L)Phe(4-CH COOH)-Lys-Gly-Trp-Met-Asp-N-methyl- 2 1 Phe-NHftowwo- PBU--LI 31 Ac-(D,L)Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N-methyl- Phe-NH 2 Desamino-Phe(4-tetrazole)Lys-Gly-T':p-Met-Asp-N-methyl- L J Phe-NH 2 Ac-(D,L)Phe(3-CH2COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Desamino-Phe(4-CH2tetrazole)-Met-Gly-Trp-Met-Asp- Phe-NH 2 Desamino-Phe(4-CH COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Desamino-Phe(4-tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH or of their pharmaceutically acceptable salts.

S°The invention is further directed to compositions for S. suppressing appetite in subjects comprising an appetite o suppressing effective amount of the peptides of the j S 20 invention, or of their pharmaceutically acceptable salts.

i Eo°. Such compositions may be prepared in a conventional way and may contain conventional carriers and/or diluents.

An "appetite suppressing effective amount" as used 'i 25 herein refers to the amount of the peptide or its pharmaceutically acceptable salt (on a weight basis) per kg of body weight of the subject which must be administered to suppress appetite. It is within the skill of the art to calculate such amounts considering the method of administration, the particular subject and the weight of the subject. See Morley, J.E. "Minireview. The Ascent of Cholecystokinin (CCK) from Gut to Brain" Life Sciences, (1982) 479.

The analogs may be administered as water soluble salts, generally as salts of alkaline metals such as sodium or potassium salts, as alkylamine salts, preferably i Y 1; I 32 diethylamine salts or as acid addition salts. The analogs of the invention can be converted to the pharmaceutically acceptable salts by known methods.

The analogs of the invention may be administered to the subject by any suitable route including, nasal, sublingual, buccal, intraperitoneal, or parenteral including intravenous, intramuscular, or transdermal.

If the analogs of the invention are administered intranasally such vehicles of administration may include foams, creams, inhalants, etc. The effective appetite suppressing amount of the analog as the active ingredient is dissolved in pharmaceutically acceptable foams or inhalant compositions suitable for intranasal administration, which 0 0, compositions are known to those skilled in the art.

'Where the peptides of the invention are administered o0 n parenterally, or intraperitoneally the appropriate amount of o 20 the analog as the active ingredient is dissolved in sterile oo o injectable solutions or suspensions. These types of solutions are well known to skilled artisans and comprise for example saline solutions, etc.

0 '0 o i 25 The present invention is also directed to the use of the peptides of the invention and their pharmaceutically 00 ~acceptable salts as therapeutically active substances, especially as appetite suppressing substances.

o 30 The peptides of the invention may be prepared using the method of solid phase synthesis as generally described by Merrifield, J. Am. Chem. Soc., 85, 2149 (1963), although other equivalent methods of chemical synthesis known in the art may also be used. Solid-phase synthesis is commenced from the C-terminal end of the peptide to be synthesized by coupling a protected a-amino acid by an amide bond to a suitable resin, benzhydrylamine (BHA), methylbenz- 33 hydrylamine (MBHA) or 4-(oxymethyl)-phenylacetamidomethyl (PAM) or or 4')-aminomethyl-3',5'-dimethoxyphenoxy]valerate (PAL). BHA, MBHA, PAM and PAL resin supports are commercially available.

All solvents used in the peptide preparations described herein, eg. methylene chloride (CH 2 C12), 2-propanol, dimethylforMaaide (DMF),:and methanol, were Burdick and Jackson "distilled in glass" grade and used without additional distillation. Trifluoroacetic acid (TFA), diisopropylethylamine (DIPEA), piperidine (PIP), dicyclohexylcarbodiimide (DCC), l-hydroxybenzotriazole (HOBt), and [benzotriazole-l-yl-oxy-tris(dimethyl) phosphonium hexafluorophosphate] (BOP) were purchased from Chemical Dynamics Corp. and were "sequential" grade purity.

1,2-ethanedithiol (EDT) was purchased from Sigma Chemical Co. and used without further purification. All protected amino acids were of the L-configuration unless otherwise indicated and were obtained from Bachem.

f In solid phase synthesis, the reactive side chain groups of the various amino acid moieties are typically protected with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the protecting group is ultimately removed. While specific protecting groups are disclosed in regard to the solid phase synthesis aspect, it should be noted that each amino acid can be protected by any protective group conventionally used for the respective amino acids in solution phase synthesis.

0 30 Purity of the protected amino acids was confirmed by thin Slayer chromatography (TLC), elemental analysis, IR, MS, NMR and optical rotation.

The following instrumentation was utilized. TLC was performed on glass backed precoated silica gel 60 F254 plates purchased from Merck using appropriate solvent systems. Detection of spots was by UV fluorescence 1 34 quenching (254 nm absorption), iodine staining, or ninhydrin spray for primary and secondary amines.

For amino acid analyses, peptides wcre hydrolyzed in 6N HC1 containing phenol at 1150 C for 24 hours in evacuated Reacti-Therm hydrolysis tubes (Wheaton Scientific Company, Milville, N.J. 08332, USA). Analyses were performed on a Beckman 121M amino acid analyzer.

High pressure liquid chromatography (HPLC) was conducted on ar laboratory data control (LDC) apparatus consisting of a Constametric I pump, a Constametric III pump, a Gradient Master solvent programmer and mixer, and a Spectromonitor III variable wavelength UV detector. Analytical HPLC ct-romatography was performed with Waters Micro Bondapack C18 on reversed phase columns (0.4 x 25 cm). Preparative o" HPLC separations were run on (2.5 x 50 cm) Partisil 10/50 ODS-3 columns, or on x 30) cm micro Bondapack SC8 column; in both cases, a pre-column of Whatman Co:Pell 18 6 20 ODS pellicular packing was used. The peptides were assembled in a stepwise manner on a solid suppcrt using a Vega 1000 peptide synthesizer. The 1000 peptide synthesizer was controlled by an Apple lie microprocessor with manual operations at steps 16 and 20 for the Boc-protocol and 7 and 10 for the Fmoc-protocol.

Boc-Phe (6.0g, 23 mmol) was coupled to the BHA resin using DCC (4.6g, 22 mmol) and HOBt (4.5g, 33 mmol) at 0 C. Loading was determined by amino acid analysis to be 0.31 mmol/g resin. Any unreacted amino groups were capped by treatment with 6 equivalents each of acetic anhydride and pyridine.

Boc-N-methyl-Phe (1.35g, 5 mmol) was coupled to the BHA resin (8g) using DCC (l.lg, 5 mmol) and HOBt (1g, 8 mmol) at 0°C. Loading was determined by amino acid analysis to be 0.30 mmol/g. Any unreacted amino groups were capped by 48 35 treatment with 6 equivalents each of acetic anhydride and pyridine.

Boc-Phe-Pam resin was purchased from Vega Biotechnologies (Tucson, Arizona, USA). The loading was 0.32 mmol/g. The PAL-linker was purchased from Biosearch (San Rafael, California, USA). The initial synthesis was started with the Boc-amino acid resin and portions of peptide resin were removed at various points of the synthetic cycle for separate analog preparations. The protocol for a typical Boc-synthetic cycle was as follows: Step Reagent Time 1 1% EDT/CH2Cl 1 x 30 sec.

2 50% TFA/CH2 Cl 1% EDT 1 x 1 min.

3 Repeat Step 1 4 50% TFA/CH Cl 1% ED 1 x 15 min.

5 CH Cl 1 x 30 sec.

o o22 oo 6 Methanol 1 x 30 sec.

o 7-8 Repeat steps 5 and 6 o 20 9 CH 2 Cl 2 x 30 sec.

a 10 8% DIPEA 2 x 2 min.

11-15 Repeat step 5-9 16 3 equiv. Boc-AA, DCC. HOBt 1 x 60 min.

S' 17 1% DIPEA 1 x 30 min.

S 25 18-19 Repeat steps 6 and 9 20-21 Repeat steps 16 and 17 if Kaiser test S" is positive 22 Methanol 1 x 30 sec.

23-24 Repeat steps 5 and 6 25 CH2C2 1 x 30 sec.

26 Methanol 2 x 30 sec.

27 CH Cl 2 3 x 30 sec.

2* 2^

_I

36 The protocol for a typical Fmoc-synthetic cycle was as follows Step 1 2 3 4 6 7 8 9 10-12 Reagent 20% piperidine/DMF 20% piperidine/DMF

DMF

CH2C12 2-propanol CH2Cl /DMF 3 equiv. Fmoc-AA, DCC, HOBt CH2C1l

DMF

Repeat steps 7, 8, 9 if Kaiser test is positive CH2Cl2

DMF

2-propanol

DMF

Time 1 x 5 min.

1 x 5 min.

2 x 1 min.

2 x 1 min.

2 x 1 min.

2 x 1 min.

1 x 60 min.

2 x 1 min.

2 x 1 min.

2 x 1 min.

2 x 1 min.

2 x 1 min.

2 x 1 min.

o 0 o 00 o o o 0 0 Q 0 1 o o Io t I (1 13 14 15 16 20 Solvents for all washings and couplings were measured to volumes of 10-20 ml/g resin. Couplings were performed using the DCC/HOBt procedure. Coupling reactions were monitored by the Kaiser ninhydrin test to determine whether coupling was complete at step 19 by the Boc-synthetic protocol or at step 9 by the Fmoc-synthetic protocol as set forth by Kaiser et al., Analytical Biochemistry 34, 595-598 (1970).

The fully assembled peptide-resins were dried under high vacuum overnight and cleaved with appropriate cleavage 30 reagents. For the Boc-synthesis the modified procedures of Tam et al. [Tetrahedron Letter, 23, 4435-4438 (1982)] were used. In brief: The peptide-resin was treated in a teflon HF apparatus (Peninsula) with HF, dimethylsulfide and p-cresol (5:13:2) for lh at 0 0 C. After evaporation to a low volume fresh anhydrous HF was distilled into the reaction vessel (18 ml) for a second treatment for 1.5 h at OOC.

After thorough evaporation, the dry resin was washed with 3 c_ i 37 volumes each of Et2 0 and EtOAc, then triturated with 4 x ml of 30% acetic acid and filtered., Lyophilization of the aqueous filtrate yielded the crude peptide.

For the Fmoc-synthesis the procedure of Mitchell et al.

Org. Chem., 43, 2845, (1978)] was used. In brief: The peptide-resin was placed into a pressure bottle, suspended in methanol, saturated with NH at -20 0 C and sealed. The 3 suspension was stirred at room temperature for 2-3 days.

After venting the excess NH 3 the PAM-resin was filtered off and washed with methanol. The filtrate was evaporated to dryness to give the crude peptide.

Preparative purification was carried out directly with the crude peptide by HPLC on a (2.3 x 30) cm micro Bondapack

S

c 8 or (2.5 x 50) cm Whatman ODS-3 column. The peptides o were applied in a minimum volume of 50% AcOH, and eluted with a slow gradient (4 hr) of 5-65%, 0.022% TFA/CH 3 CN, at Sa flow rate of 8.0 ml/min. Fractions were collected at 3 S° 20 minute intervals and cuts were made after inspection by S analytical HPLC. Fractions, judged to be greater than 97% pure, were pooled and lyophilized.

°o o Purity of the individual peptides was checked by HPLC o 25 and determined to be 99% in all cases. Amino acid analyses of the individual peptides were performed and the expected values were obtained in each case. N.M.R. and M.S.

were also performed on the analogs confirming the chemical integrity of the peptides.

The separation of peptides containing amino acids into their optically pure enantiomers was achieved by preparative HPLC on (2.3 x 30) cm micro Bondapack C 18 column from E.S. Industries using CH 3 CN/0.01M NH4OAC.

The chirality of the amino acids were determined by the method of Bayer et al. Frank, W. Woiwode. G. Nicholson 38 and E. Bayer, Liebig Ann. Chem. 354, (1981)] using glass capillary gas chromatography.

The present invention will be further described in connection with the following examples which are set forth for the purposes of illustration only.

For the preparation of the compounds described in Examples 1-36, melting points were taken on a Buchi 510 melting point apparatus and are uncorrected. Preparative high pressure liquid chromatography (HPLC) was performed on silica gel Pre-Pak 500 cartridges using a Waters Associates Prep LC 500A. Dry dichloromethane was distilled from P205, DMF was dried over Linde 3A molecular sieves and triethylamine was distilled from calcium hydride.

o;a, Concentration refers to evaporation under aspirator vacuum «o using a Buchi rotary evaporator.

4 I I I .0 t I 2 _i i 39 EXAMPLE 1 Preparation of (S)-4-rf(trifluoromethyl)sulfonylloxylalpha-[r(1,1-dimethylethoxy)carbonyllaminolbenzenepropanoic acid methyl ester A solution of 19.23 g (0.065 mol) of (S)-4-hydroxyalpha[[(1,1-dimethylethoxy)carbonyl]amino]benzenepropanoic acid methyl ester and 24.0 g (0.067 mol) of N-phenyl-Ntrifluoromethylsulfonyl-1,1,1-trifluoromethane sulfonamide in 175 ml of dry dichloromethane was cooled in an ice bath and 9.7 ml (0.70 mol) of triethylamine was added over three minutes. The resulting mixture was held at o 0 C for 1 hour and allowed to warm to room temperature over 1 hour. The reaction mixture was diluted with 500 ml of ether and washed successively with water (1 X 100 ml), 1 N sodium hydroxide on solution (2 X 100 ml), water (1 X 100 ml), and saturated sodium chloride solution (1 X 100 ml). The organic phase was dried over magnesium sulfate and concentrated to an oil which was purified by preparative liquid chromatrgraphy a using silica gel cartridges on a Waters Prep 500 chromatograph, eluting with 20% ethyl acetate-hexane. The pure fractions were combined and evaporated to give 26.78 g 0 of (S)-4-[[(trifluoromethyl)sulfonyl]oxy]-alpha- 25 [[(1,1-dimethylethoxy)carbonyl]aminolbenzenepropanoic acid methyl ester as a colorless oil which crystallized on i. t, standing, melting point (mp) 48-49 0

C.

EXAMPLE 2 Preparation of (S)-alpha-[[(1,1-dimethylethoxy)carbonyllaminol-4-(2-propenyl)benzenepropanoic acid methyl ester Argon was passed through a solution of 7.0 g (0.0164 mol) of (S)-4-[[(trifluoromethyl)sulfonyl]oxy]-alpha-[[(1,1dimethylethoxy)carbonyljaminojbenzenepropanoic acid methyl ester, 5.7 g (0.0165 mol) of allyltributyl tin, and 1.42 g ~L 40 (0.04 mol) of lithium chloride in 50 ml of dimethylformamide for 10 minutes and 210 mg (0.0003 mol) of i bis(triphenylphosphine)palladium dichloride was added. The bath temperature was raised to 90-95 0 C for 40 minutes and the mixture was allowed to cool. The mixture was diluted with ether and washed with water and saturated sodium i chloride solution and dried over magnesium sulfate. The i residue obtained after filtration and evaporation was purified by preparative liquid chromatography using silica gel cartridges on a Waters Prep 500 chromatograph, eluting with 10% ethyl acetate-hexane to give 4.83 g of (S)-alpha-[[(l,l-dimethylethoxy)carbonyl]amino]-4-(2propenyl)benzenepropanoic acid methyl ester, mp 56-59 0

C.

i i 15 Example 3 I oo Preparation of (S)-4-carboxymethyl-alpha-[ (1,1- O dimethylethoxy)carbonyllaminolbenzenepropanoic acid methyl i ester II o 0 f I To a solution of 4.00 g (0.0125 mol) of (S)-alpha- S[[(1,l-,-.imethylethoxy)carbonyl]amino]-4-(2-propenyl)benzenepropanoic acid methyl ester in 80 ml of carbon tetrachloride and 80 ml of acetonitrile was added a solution S 25 of 8.00 g (0.037 mol) of sodium metaperiodate in 200 ml of water. The two phase mixture was stirred mechanically and l 0 0.2 g (0.0001 mol) of ruthenium trichloride hydrate was K added. The resulting dark mixture was stirred at room temperature for 1 hour and was diluted with 500 ml of 30 dichloromethane. The layers were separated and the organic layer was washed with water and was dried over magnesium sulfate. Filtration and evaporation gave 3.84 g of a dark oil. This oil was dissolved in 120 ml of t-butyl alcohol and 40 ml of 2-methyl-2-butene and a solution of 12.6 g of sodium chlorite and 12.6 g of sodium dihydrogen phosphate in ml of water was added. The resulting mixture was stirred mechanically for 2 hours. The mixture was diluted with L ,i i 41 500 ml of ether, the layers were separated, the organic phase was washed with 100 ml portions of water, 10% sodium thiosulfate and saturated sodium chloride solution and was dried over magnesium sulfate. The residue obtained after filtration and concentration was chromatographed over 150 g of silica gel, eluting with 40:59:1 ethyl acetate-hexaneacetic acid. The earlier fractions contained 0.631 g of a mixture from which (S)-4-carboxy-alpha-[[(1,1dimethylethoxy)carbonyl]amino]benzenepropanoic acid methyl ester was obtained by crystallization from ether-hexane, mp 101-104 0 C, [a] D +4.00 (ethanol). The later fractions were combined, diluted with toluene and evaporated to remove traces of acetic acid and were evacuated under high vacuum for 72 hours to give 3.078 g of (S)-4-carboxymethyl-alpha- [[(1.1-dimethylethoxy)carbonyl]amino]benzenepropanoic acid i methyl ester as a colorless oil, dicyclohexylamine salt, mp 141-143 0 C, [a]D+7.

25 (ethanol).

EXAMPLE 4 o Preparation of (S)-alpha-[[(1,1-dimethylethoxy)carbonyl]aminoj-4-[2-(1,1-dimethylethoxy)-2-oxoethyl]benzenepropanoic acid methyl ester 25 a. A solution of 2.85 g of (S)-4-carboxymethyl)alpha- [E(1,1-dimethylethoxy)carbonyl]amino]benzenepropanoic acid methyl ester in 12 ml of t-butyl alcohol was treated with 2.06 g of dicyclohexylcarbodiimide followed by 0.13 g of 4-pyrrolidinopyridine. A white precipitate formed after a 30 few minutes and the reaction mixture was allowed to stir for 0, 18 hours. The mixture was filtered, and the solid washed with ether. The ether extracts and the filtrate were combined and washed with IN hydrochloric acid, water, and saturated sodium bicarbonate solution and were dried over magnesium sulfate. The residue obtained after filtration and evaporation was chromatographed over 150 g of silica gel, eluting with 20% ethyl acetate-hexane to afford 1.613 g I I_ 42 of (S)-alpha-[[(1,1-dimethylethoxy)carbonyllamino]-4-[2- (1,1-dimethylethoxy)-2-oxoethyl]benzenepropartnoic acid methyl ester as a colorless oil, [a]D +6.07 (ethanol).

b. A solution of 3.50 g of (S)-4-carboxymethyl)-alpha- [[(1,1-dimethylethoxy)carbonyljamino]benzenepropanoic acid methyl ester in 40 ml of dry toluene and 10 ml of dimethylformamide di-t-butyl acetal was heated to a bath temperature of 80 0 C for 4 hours. After cooling, the mixture was diluted with 100 ml of ether and was washed with water and saturated sodium chloride solution and was dried over magnesium sulfate. The residue obtained after filtration and evaporation was chromatographed over 150 g of silica gel, eluting with 20% ethyl acetate-hexane to give 2.663 g of (S)-alpha--[(1,l-dimethylethoxy)carbonyl]amino] -4-[2-(1,1-dimethylethoxy)2-oxoethyl]benzenepropanoic acid ~methyl ester.

EXAMPLE Preparation of (S)-alpha-f[(1,1-dimethvlethoxy)carbonylI aminol-4-f2-(l1,1-dimethylethoxy)-2-oxoethyllbenzenepropanoic acid 25 A solution of 1.471 g of (S)-alpha-[[(1,1-dimethylethoxy)carbonyl]amino]-4-[2-(1,1l-dimethylethoxy)-2-oxoethyljbenzenepropanoic acid methyl ester in 25 ml of methanol and ml of 1 N sodium hydroxide solution was stirred at room I temperature for 2 hours. The mixture was acidified with a 30 slight excess of hydrochloric acid, was diluted with 100 ml of ether, and was washed with water and saturated sodium chloride solution. The residue obtained after filtration and evaporation was chromatographed over 100 g of silica gel, eluting with 40:59:0.5 ethyl acetate-hexane-acetic acid. The product containing fractions were combined, evaporated, diluted with toluene and evaporated finally under high vacuum to give 1.105 g of (S)-alpha-[[(1,1- L- 43 -dimethylethoxy)carbonyl]amino]-4-[2-(l1,-dimethylethoxy)- -2-oxoethyl]benzenepropanoic acid [aID 21.41 (ethanol).

EXAMPLE 6 Preparation of 5-(4-methylphenyl)-2-(l,l-dimethylethyl)- 2H-tetrazole A solution of 5-(4-methylphenyl)-2H-tetrazole [Finnegan, W.G. et al., J. Am. Chem. Soc. 80, 3908, (1958)] (19.9 g, 0.12 mol), t-butanol (19.5 g, 0.26 mol) and concentrated sulfuric acid (5.84 g, 0.06 mol) in trifluoroacetic acid (122 ml) was stirred at room temperature (3 h) and then diluted with ethyl acetate (250 ml). The mixture was washed sequentially with water (2 x 50 ml), 10% aqueous sodium hydroxide until washings were basic, water (2 x 30 ml), and then dried over sodium sulfate (Na 2

SO

4 and filtered.

0o Following removal of the solvents in vacuo, the resultant o re 0 o oil was purified by HPLC on silica gel eluting with 20:1 S 20 mixture of hexanes-ethyl acetate to provide 17.5 g of 5-(4-methylphenyl)-2-(l,1- dimethylethyl)-2H-tetrazole as a clear oil, bp. 120-122 0 C (0.3 mm Hg).

EXAMPLE 7 0*0 Preparation of benzyl bromide derivatives 00 N-Bromosuccinimide (NBS) (35.8 g, 0.20 mol) was added in several portions to a solution of 1,1-dimethylethyl 30 4-methylbenzoate (prepared by the reaction of the respective acid chloride with t-butanol.) (38.Og, 0.20 mol) and benzoyl peroxide (0.36 g, 1.5 mmol) in carbon tetrachloride (1200 ml) at vigorous reflux. Another portion of benzoyl peroxide (0.36 g, 1.5 mmol) was added just before the final addition of NBS. After the foaming subsided, the reaction mixture was cooled to room temperature, washed with water (2 x 60 ml) and dried over magnesium sulfate. Filtration L

I_

~CI- U CII~ -U Ilp i 44 and concentration provided an oil that was purified by HPLC on silica gel eluting with a mixture of hexanes and ethyl acetate to give 1,1-dimethylethyl 4-bromomethylbenzoate (21.4 g) in 40% yield.

Following the above procedure, the analogs listed below were prepared.

a. From 1,1-dimethylethyl 3-methylbenzoate (40.0 g, 0.21 mol) there was obtained 1,1-dimethylethyl 3-bromomethylbenzoate (23.0 g, 0.09 mol) in 41% yield.

b. From 1,1-dimethylethyl 2-(4-methylphenyl)acetate (25.0 g, 0.12 mol) there was obtained 1,1-dimethylethyl 2-(4-bromomethylphenyl)acetate (10.9 g, 0.04 mol) in 32% 0 0o yield.

o c. From 5-(4-methylphenyl)-2-(1,1-dimethylethyl)- 2H-tetrazole (18 g, 0.08 mol) there was obtained o a O 20 5-(4-bromomethylphenyl)-2-(1,l-dimethylethyl)-2H-tetrazole (17.4 g, 0.06 mol), in 71% yield.

d. From 1,1-dimethylethyl 2-(4-methylphenyl)-2,2difluoroacetate (8.0 g, 0.03 mol) there was obtained 25 1,1-dimethylethyi 2-(4-bromomethylphenyl)-2,2-difluoroacetate (7.7 g, 0.024 mol) in 73% yield.

EXAMPLE 8 Preparation of 5-[(4-bromophenyl)methyl]-2H-tetrazole 4-Bromophenylacetonitrile (30.0 g, 0.15 mol), sodium azide (10.9 g, 0.17 mol) and ammonium chloride (8.9 g, 0.17 mol) were heated in DMF (300 ml) at 90 0 C for 2 days.

After concentration, water (200 ml) was added to the residue, the mixture was basified with 1 M NaOH (170 ml) and washed with ether (2 x 100 ml). Acidification of the 45 aqueous layer with IN HC1 and collection of the precipitate by suction filtration produced the crude product. This was purified by recrystallization from ethanol to provide 5-[(4-bromophenyl)methyl]-2H-tetrazole (17.2 g, 0.07 mol) in 44% yield, mp. 173-175 0

C.

EXAMPLE 9 Preparation of 5-[(4-bromophenyl)methyll-2-(1,1dimethylethyl)-2H-tetrazole A mixture of 5-[(4-bromophenyl)methyl]-2H-tetrazole (18.5 g, 77 mmol), t-butanol (11.4 g, 150 mmol), trifluoroacetic acid (76 ml, 1.0 mol) and concentrated sulfuric acid (3.8 g, 39 mmol) was stirred for 24 h and then partitioned between ethyl acetate (250 ml) and water (100 ml). The organic layer was washed with water (4 x 100 ml) and 1 M NaOH (2 x 100 ml) and was dried over sodium sulfate. Filtration and removal of the solvent in vacuo produced a -lid that was recrystallized from hexane to give 5-[(4-bromophenyl)methyl]-2-(l,1-dimethylethyl)-2H-tetrazole.

(12.3 g, 47 mmol) in 54% yield, mp. 69-70 0

C.

EXAMPLE Preparation of 4-[[2-(1,l-dimethylethyl)-2H-tetrazolacid To a deoxygenated mixture of 5-[(4-bromophenyl)methyl] -2-(1,1-dimethylethyl)-2H-tetrazole (12.0 g, 41 mmol), triphenylphosphine (11.0 g, 42 mmol), tributylamine (8.4 g, mmol), t-butanol (70 ml) and water (45 ml) was added bis(triphenylphosphine)palladium dichloride (1.0 g, 13 mmol). This mixture was placed in a glass lined autoclave and maintained under carbon monoxide (200 psi) at 100 0 C for 2 days. The reaction mixture was diluted with dichloromethane (500 ml), washed with water (3 x 100 ml) and -46 dried over magnesium sulfate. Filtration and concentration produced the crude product mixture. This was purified by flash chromatography on silica gel using a 4:1 mixture of hexane and ethyl acetate to remove the less polar impurities and 5% acetic acid in hexane-ethyl acetate to elute (l1,-dimethylethyl)-2H-tetrazole-5-yl]methyl]benzoic acid (4.2 g, 16 mmol) in 49% yield, mp. 139-140 0

C.

EXAMPLE 11 Preparation of 4-[[2-(1,1-dimethylethyl)-2H-tetrazol-5-yllmethyl]benzyl alcohol Diisobutylaluminum hydride (48 ml of a 0.8 M solution in toluene, 38 mmol) was added dropwise to a mixture of 4-[[2-(1,1-dimetethylethy)-2H-tetrazole-5-yl]methyl]benzoic acid (2.5 g, 9.6 mmol) in toluene (250 ml) at 0°C and the 4 44 4 mixture was then warmed to room temperature. After stirring overnight, the reaction was quenched at 0°C by careful addition of a mixture of water and THF (8 ml, 10% NaOH ml) and a final portion of water (15 ml). After the addition of ether (200 ml) and stirring at room temperature for 2 hours, tle white precipitate was removed by filtration and the filtrate was concentrated. The residue was purified by HPLC on silica gel eluting with a 7:3 mixture of hexane-ethyl acetate to give 4-[[2-(1,1-dimethylethyl)- (1.6 g, 6.7 mmol) in yield, mp. 65-66 0

C.

EXAMPLE 12 Preparation of 5-[[4-(Bromomethyl)phenyllmethyl]-2- (1,1-dimethylethyl)-2H-tetrazole Triphenylphosphine (6.5 g, 25 mmol) was added to a solution of 4-[[2-(1,1-dimethylethyl)-2H-tetrazol-5-yl]methyl]benzenemethanol (3.0 g, 12.4 mmol) and carbon

-A

PAI~I" i b 47 tetrabromide (8.3 g, 25 mmol) in diethyl ether (150 ml).

After 3 hours the reaction mixture was concentrated and the residue purified by chromatography on silica gel eluting with a 9:1 mixture of hexane-ethyl acetate to give 5-[[4-(bromomethyl)phenyl]methyl]-2-(1,1-dimethylethyl)- 2H-tetrazole (2.4 g, 7.8 mmol) in 63% yield, mp. 73-74oC.

EXAMPLE 13 Preparation of benzyl phenylalaninate-benzophenone imine derivatives Tetrabutyl ammonium sulfate (3.0 g, 9 mmol) was added to a biphasic mixture consisting of 10% aqueous sodium hydroxide (75 ml), 2-[(diphenylmethylene)amino]acetic acid Sbenzyl ester (3.05 g, 9.3 mmol) and 1,1-dimethylethyl 1 4-bromomethylbenzoate (3.0 g, 9 mmol) in dichloromethane (58 ml). After several hours of vigorous stirring at room temperature, the reaction mixture was diluted with ether (300 ml) and the layers were separated. The organic layer was washed with water (3 x 30 ml), dried (K 2

CO

3 and filtered. Concentration provided an oil that was purified by HPLC on silica gel (the columns were pre-treated with o o triethylamine/hexanes, ethyl acetate and finally hexanes) o\o 25 eluting with a mixture of hexanes-ethyl acetate to give a rac.- 2 -[(diphenylmethylene)amino]-3-[4-[(1,1-dimethylethoxy) carbonyl]phenyl]propanoic acid benzyl ester, (1.7 g, 3 mmol) in 36% yield.

Following the above procedure, the analou, listed below S0 were prepared.

a. From 1,1-dimethylethyl 3-bromomethylbenzoate (10.0 g, 37 mmol) there was obtained rac.-2-[(diphenylmethylene) amino]-3-[3-[(1,1-dimethylethoxy)carbonyl]phenyl]propanoic acid benzyl ester, (9.4 g, 18 mmol) in 49% yield.

48 b. From 1,1-dimethylethyl 4-bromomethyiphenylacetate (3.1 g, 11 mmol) there was obtained rac.-2-[(diphenylmethylene)amino]-3-[4-2-(1,1.dimethylethoxy>2oxoethyl] phenylipropanoic acid benzyl ester, (2.9 g, 5 mmol) in yield.

c. From 5-(4-bromomethylphenyl)-2-(1.1-dimethylethyl) -2H-tetrazole (16.0 g, 54 mmol) there was obtained rac.-2- [(diphenylmethylene)amino-3-[4-[5-[(2-( 1,1dimethylethyl) -2H-tetrazoyl)jiphenylpropanoic acid benzyl ester, (10.9 g, 32 mmol) in 59% yield.

d. From S-(4-bromomethylphenylmethyl)-2-(1,1dimethylethyl)-2Htetrazole (2.0 g, 6.5 mmol) there was obtained rac.-2-[(diphenylmethylene)aminoj-3-[4-[52 (1,1-dimethylethyl)-2H.tetrazoylj ]methyl phenylI propanoic acid benzyl ester, (1.9 g, 3.3 mmol) in 50% yield.

EXAMPLE 14 Preparation of rac.-2-r(diphenylmethylene)aminol-3-r4- Fl1-difluoro-r2-(1,1-dimethylethoxy)-2-oxoethyllphenyl1 propanoic acid benzyl ester Lithium diisopropyl amide (8.8 mmol) was generated in ml of TEF at 0 0 C and chilled to -78 0 C. A solution of 2-[(diphenylmethylene)amino]acetic acid benzyl ester (2.9 g, 8.8 mmol) in THF (5 ml) was added over 5 minutes followed by the dropwise addition of hexamethylphosphoramide (1.6 g, 8.8 mmol) and the mixture was stirred for 15 min. A solution of 1,1-dimethylethyl 2-(4-bromomethylphenyl)-2,2difluoroacetate (2.7 g, 8.0 mmol) in 5 ml THF was added slowly and the reaction mixture was warmed over the course of 2 hours to room temperature. The mixture was partitioned between saturated aqueous NH 4 C1 (50 ml) and ether mi). The aqueous layer was extracted with additional ether (2 x 30 ml), and the combined organic layers were washed I- 49 with brine (20 ml), and dried (MgSO 4 Filtration and concentration yielded an oil that was purified by flash chromatography on silica gel (pretreated with triethylamine in hexanes) eluting with a 9:1 mixture of 5 hexane-ethyl acetate. This provided rac.-2-[(diphenylmethylene)amino]-3-[4-[1,1-difluoro-[2- (1,1-dimethylethoxy)-2-oxoethyl]phenyl] propanoic acid benzyl ester (3.2 g, 5.6 mmol), as a colorless oil in 71% yield.

EXAMPLE Preparation of benzyl phenylalaninate p-toluenesulfonic acid salt derivatives A solution of rac.-2-[(diphenylmethylene)amino]-3-[4- [(1,1-dimethylethoxy)carbonyljphenyl]propanoic acid benzyl S. ester (11.5 g, 22 mmol) and p-toluenesulfonic acid 02 monohydrate (4.18 g, 22 mmol) in a 10/1 mixture of acetonitrile and water (640 ml) was stirred for 2 h at room temperature. Concentration provided a crude solid product which was recrystallized from ethanol-ether to give rac.-2amino-3-[4-[(1,1-dimethylethoxy)carbonyl]phenyl]propanoic acid benzyi ester p-toluenesulfonic acid salt, (8.1 g, mmol) in 67% yield, mp. 154-157 0

C.

Following the previous procedure, the analogs listed below were prepared.

a. From rac.-2-[(diphenylmethylene)aminoj-3-[3-[(1,1dimethylethoxy)carbonyl]phenyl]propanoic acid benzyl ester (9.4 g, 18 mmol) there was obtained rac.-2-amino-3-[3-[(1,1dimethylethoxy)carbonyl]phenyljpropanoic acid benzyl ester p-toluenesulfonic acid salt which was carried on to the next step without purification.

b. From rac.-2-[(diphenylmethylene)amino]-3-[4-[2-(1,1.

dimethylethoxy)-2-oxoethyl]phenyl]propanoic acid benzyl ester (6.9 g, 13 mmol) there was obtained rac.-2--amino-3-[4l-dimethylethoxy)--2-oxoethyljphenyljpropanoic acid benzyl ester p-toluenesulfonic acid salt, (6.1 g, 11 mmol) in 87% yield, mp. 139-141 0

C.

c. From rac.-2-[ (diphenylmethylene)aminoj-3-[4-[l-dimethylethyl)-2H-tetrazoyljphenyljpropanoic acid benzyl ester (8.0 g, 15 mmol) there was obtained rac.-2amino-3-[4-[5-[2-(l,1dimethylethyly2H-tetrazoyl]Jphlenyl]y propanoic acid benzyl ester p-toluenesultonic acid salt, (6.6 g, 12 mmol) in 81% yield, rnp. 204-205 0

C.

d. From rac.-2-[(diphenylmethylene)amino-34jl,1difluoro-[2-(l, 1-dimethylethoxy)-2-oxoethyljphenyljpropanoic acid benzyl ester (3.2 g, 5.6 mmol) there was obtained rac.-2-amino-3-[4[,1difluoro[2-(1,1dimethylethoxy)2 oxoethyl]phenyllpropanoic acid benzyl ester p-toluenesultonic acid salt, (2.3 g, 3.9 mmol) in 70% yield, mp. 145-149 0

C.

e. From rac.-2-[(diphenylmethtylene)amino-3-[4.

2 -(l,1-dimethylethyl)-2R..tecrazoyljmethyl]phenylppropanoic acid benzyl ester (4.4 g, 7.9 mmol) there was obtained rac.-2--amino-3-[4-t 1-dimethylethyl)a. 2H-tetrazoyl)Jmethyljphenyljpropanoic acid benzyl ester p-toluenesulfonic acid salt, (3.0 q, 5.3 mmol) in 67% yield, mp. 151--154 0

C.

EXAMPLE 16 Preparation of rac.-benzyl N-acetyllhenrlalaninate derivatives Acetic anhydride (1.0 ml. 10.8 mmol) and triethylamine (2.4 g, 24 mmol) were added sequentially to a solution of r ~I ~~YLI^L-II-_IIY -I S1 rac.-2-amino-3-[4-[(1,1-dimethylethoxy)carbonyl]phenyl] propanoic acid benzyl ester p-toluenesulfonic acid salt g, 9 mmol) in dichloromethane (100 ml) at 0 0 C. After 2 h, the reaction mixture was diluted with dichloromethane (250 ml) and washed with 1 N HC1 (2 x 20 ml), saturated aqueous sodium bicarbonate (2 x 20 ml) and dried (Na 2

SO

4 Filtration and concentration provided the crude product which was purified by HPLC on silica gel eluting with a mixture of hexanes and ethyl acetate to give rac.- 2-acetamido-3-[4-[(1,1-dimethylethoxy)carbonyl]phenyl]propanoic acid benzyl ester (3.3 g, 8 mmol) in 91% yield.

Following the above procedure, the analogs listed below were prepared.

oa a. From rac.-2-amino-3-[3-[(1,1-dimethylethoxy)oDe 0 carbonyl]phenyl]propanoic acid benzyl ester n p-toluenesulfonic acid salt obtained as the crude product in the hydrolysis of the corresponding diphenylmethyl imine, there was obtained rac.-2-acetamido-3-[3-((1,1-dimethylethoxy)carbonyl]phenyl]propanoic acid benzyl ester as an oil.

C b. From rac.-2-amino-3-[4-i2-(1,1-dimethylethoxy)-2- 25 oxothyl]phenyllpropanoic acid benzyl ester p-toluenesulfonic acid salt (5.9 g, 11 mmol) there was obtained rac.-2-acetamido-3-[4-[2-(1,1-dimetylethoxy)-2oxoethyl]]phenyl]propanoic acid benzyl ester, (3.1 g, 8.4 mmol) in 76% yield.

4c. From rac.-2-amino-3-[4-[5-[ 2 -(l1,1-dimethylethyl)- 2H-tetrazoyl]Iphenyl]propanoic acid benzyl ester p-toluenesulfonic acid salt (2.0 g, 3.8 mmol) there was obtained rac.-2-acetamido-3-[ 4 2 -(1,1-dimethylethyl)- 2H-tetrazoyl]phenyl]propanoic acid benzyl ester, (1.4 g, 3.3 mmol) in 87% yield.

d. From rac.-2-amino-3-[4-[1,1--difluoro-[2-(I,1dimethylethoxy)-2-oxoethyl]]phenyl]propanoic acid benzyl ester p-toluenesulfonic acid salt (2.0 g, 3.4 mmol) there was obtained rac.-2-acetamido--3-[4-L1,1-difluoro-[2-(1,1 dimethylethoxy)-2-oxoethyl]]phenyl]propanoic acid benzyl ester, (1.2 g, 2.7 mmol) in 80% yield, mp. 98-100 0 C (ethyl acetate/hexanes).

e. From rac.-2-amino-3-t4-[[5-[2-(1,1-dimethylethiyl)acid benzyl ester p -toluenesulfonic acid salt (4.3 g, 7.4 mmol) there was obtained rac.-2-amino-3-[4-[t5-[2-(1,1-dimethylethyl)-2Htetrazoyl]]methyl]phenyl]propanoic acid benzyl ester, g, 4.6 mmol) in 63% yield, mp. 113-114.51C (ethyl acetate/hexanes).

A

EXAMPLE 17 Preparation of N-acetylphenylalanine derivatives A suspension of rac.-2-acetamido-3-[4--[(l,1-dimethylethoxy)carbonyl]phenyljpropanoic acid benzyl ester (3.3 g, 8 mmol) and 10 Pd/C (320 mg) in ethanol (100 ml) was blanketed with hydrogen (1 atm~) at room temperature. Upon the consumption of 1 equivalent of hydrogen, the mixture was filtered through celite. The celite pad was washed with ethanol (50 ml) and the filtrates were combined and concentrated. The resultant solid was purified by recrystallization from ethyl acetate/hexane to give rac.-2-acetamido-3-[4-[(1,1-dimethylethoxy)carbonyl]phenyl] propanoic acid, (2.3 g, 7.4 mmol) in 89% yield, mp.

187-188 0

C.

Following the above procedure, the analogs listed below were prepared.

53 a. From rac.-2-acetamido-3-[3-[(1,1-dimethylethoxy)carbonyl]phenyljjpropanoic acid benzyl ester (3.1 g, 7.8 mmol) there was obtained rac.-2-acetamido-3--[3-[(1,1dimethylethioxy)carbonyljphenylJpropanoic acid, (1.8 g, 5.8 ramol) in 75% yield, mp. 164-166 0 C (ethyl acetate/hexanes).

b. From rac.-2-acetamido-3-[4-(-2--(1,1-dimethylethoxy)-2--oxoethyljphenyl~propanoic acid benzyl ester (2.6 g, 6.5 mmol) there was obtained rac.-2--acetamidol-dimethylethoxy)--2-oxoethyl]phenyl]propanoic acid, (1.9 g, 5.9 mmol) in 91% yield, rap. 170-172 0 C (ethyl acetate/hexanes).

C. From rac.-2-acetamido-3-[4-[3-[2-(1,1-dimethylethyl)-2H-tetrazoyl]jphenyl]propanoic acid benzyl ester 0 (3.2 g, 7.6 mmol) there was obtained rac.-2-acetamido-3- -(4-[5-[(2-(1,l-dimethylethyl)-2H-tetrazoylj]phenyljpropanoic 0 acid, (2.2 g, 6.5 ramol) in 86% yield, rap. 206-207 0 C (ethyl C C 20 acetate/hexanes).

d. From rac.-2-acetamido-3--f4-111,1-difluoro-[2-(1,1 dimethiylethoxy) 2-oxoethylj phenyl jpropanoic acid benzyl ester (1.1 g, 2.5 ramol) there was obtained rac.-2--acetamido- 3-[4-[1,l-difluoro[2-(1,1-dimnethiylethoxy)-2-oxoethyljphenyl] propanoic acid, (0.6 g, 1.7 mmol) in 69% yield, rap. 149-150.5 0 C (acetonitrile).

e. From rac.-2-acetamido-3-[4-[[5-[2-(1,1-dimethylethyl)-2H-tetrazoyl]Jmethyljphenyljpropanoic acid benzyl ester (2.0 g, 4.6 mmol) there was obtained rac.-2-acetamido-3-[4-[[5-[2-(1,1-dimethylethyl)- 2H-tetrazoyl]Jmethyljphenyl] propanoic acid, (1.2 g, mmol) in 76% yield, mp. 193-194.5 0 C (acetonitrile).

s~ lrrrg-- l ~I.~~J~IXr~k~F 54 EXAMPLE 18 Preparation of (S)-2-r(1,l--dimethylethoxy)carbonliaminol-3-[4-rf(trifluoromethl)sulfonylloxylphenyllpropanoic acid diphenylmethyl ester Triethylamine (4.0 g, 39 mmol) was added dropwise to a suspension of (S)-2-[[(1,1-dimethylethoxy)carbonyl]amino]-3-(4-hydroxyphenyl)propanoic acid diphenyimethyl ester (17.0 g, 38 mmol) and N-phenyl-N-trifluorosulfonyl -1,1.1-trifluoromethyl sulfonamide (13.6 g, 38 mmol) in dichloromethane (170 ml) at 0 0 C. After 1 hour the reaction mixture was warmed to room temperature and washed with 1 N sodium hydroxide (2 x 25 ml), saturated aqueous sodium bicarbonate (2 x 25 ml) and water (2 x 25 ml). The organic layer was dried (K 2 C0 3 filtered and concentrated.

Recrystallization of the solid from hexane gave (S)-2-[[(1,1-dimethylethoxy)carbonyljamino]-3-[4-[L( trifluoromethyl)sulfonylJoxy]phenyl3propanoic acid 20 diphenylmethyl ester (13.7 g, 24 mmol) in 62% yield.

mp. 110-112 0 C, JD 4 23 0 (0.12% in ethanol).

04 0 00 0 0o 00 0 0 00 4 a EXAMPLE 19 Preparation of (S)-2-tF(1,1-dimethyletoxy)carbonyllamino]-3-[4-[3-(l,l-dimethlethoxy)3-oxo-l-p openyllphenyll propanoic acid diphenylmethyl ester 4 4 Bis(triphenylphosphine) palladium dichloride (120 mg, 0.2 mnol) was added to a deoxygenated mixture of (S)-2-[[(1,1-dimethylethoxy)carbonyl~amino]-3-[4- [[(trifluoromethyl)sulfonyl]oxy]phenyl]propanoic acid diphenylmethyl ester (3.0 g, 5.2 mmol), t-butyl acrylate ml, 10.2 mmol) and triethylamine (4.0 ml, 30.0 mmol) in DMF (50 ml) and the well-stirred suspension was then heated at 90 0 C (24 The solvent was removed in vacuo and the resulting residue was purified by flash chromatography on c_ silica gel eluting with a mixture of dichloromethane and hexanes to give the (S)-2-[[(1,1-dimethylethoxy) carbonyljamino]-3-[4-[3-(1,1-dimethylethoxy)-3-oxo-1propenyl]phenyl]propanoic acid diphenylmethyl ester (1.6 g, 2.8 mmol) in 54% yield, mp. 135-137 0 C, [a]D -5.350 (0.97% in ethanol).

EXAMPLE Preparation of (S)-2-fr(1,1-dimethylethoxy)carbonyllamino]-3-E4-f3-(1,1-dimethylethoxy)-3-oxopropyllphenyllpropanoic acid 2 -[[(1,1-Dimethylethoxy)carbonyl]amino]-3-[4-[3- (1,1-dimethylethoxy)-3-oxo-l-propenyl]phenyljpropanoic acid *,diphenylmethyl ester (1.6 g, 2.8 mmol) and 10 Pd/C O: (300 mg) in t-butanol (20 ml) was blanketed with hydrogen (1 atm). To prevent the solvent from freezing, a warm water bath (40 0 C) was used to heat the reaction mixture. Upon the consumption of 2 equivalents of hydrogen, the mixture was filtered through celite. The celite pad was washed with ethanol (50 ml) and the filtrates combined and concentrated. The residue was purified by recrystallization from hexanes to give (S)-2-[[(1,1-dimethylethoxy) carbony1]amino]-3-[4-[3-(1,1-dimethylethoxy)3-oxopropyl]phenyl]propanoic acid (1.0 g, 2.6 mmol) in 94% yield, mp.

88.5-90 0 C, [c]D+ 2 1 2 0 (0.90% in ethanol).

EXAMPLE 21 Preparation of benzyl 2-(4-bromophenyl)acetate A mixture of 2-(4-bromophenyl)acetic acid (50 g, 0.23 mol), benzyl alcohol (26 ml, 0.25 mol) and p-toluenesulfonic acid (0.3 g) in 200 ml toluene were heated at reflux with the azeotropic removal of water (15 The yellow solution was washed with water (250 ml), saturated aqueous sodium 56 bicarbonate (250 ml) and then dried (MgSO4 4 Concentration provided a light yellow solid that was recrystallized from cold hexanes to give benzyl 2-(4-bromophenyl)acetate (48.5 g, 0.16 mol) in 69% yield, mp. 47.5-48 0

C.

EXAMPLE 22 Preparation of benzyl (E)-2-[4-r3-(1,1-dimethylethoxy)-3oxo-1-propenyllphenyllacetato A solution of benzyl 2-(I-bromophenyl)acetate (33.5 g, 0.11 mol), t-butyl acrylate (31.6 ml, 0.22 mol) and triethylamine (85 ml, 0.64 mol) in DMF (1000 ml) was deoxygenated with argon, whereupon bis(triphenylphosphine)palladium dichloride (5.1 g, 8.3 mmol) was added. After o heating at 75 0 C for 12 hours the solvent was removed a :in vacuo providing a brown residue that was purified by chromatography on silica gel using a mixture of hexanes and ethyl acetate to give benzyl thylethoxy)-3-oxo-1-propenyl]phenyl]acetate as a pale yellow oil (23.3 g, 0.07 mol) in 60% yield.

EXAMPLE 23 0 o Preparation of 1,1-dimethylethyl 4-(carboxymethyl)phenylpropanoate A mixture of benzyl (E)-2-[4-[3-(1,1-dimethylethoxy)-3- 30 oxo-1-propenyl]phenyl]acetate (11.6 g, 33.0 mmol) and 0 Pd/C (0.5 g) in ethanol (120 ml) was stirred under H2 for 3 hours at 1 atm. Filtration of the mixture through a pad of Celite, followed by concentration gave a colorless oil that was recrystallized from a mixture of ethyl acetate and hexanes to give 1,1-dimethylethyl-4-(carboxymethyl)phenyl propanoate (7.5 g, 28.4 mmol) in 86% yield, mp. 55.5-56.5 0

C.

57 EXAMPLE 24 Preparation of 1,1-dimethylethyl 3-[4-(2-benzyloxy-2-oxoethyl)phenyl]propanoate To a stirred suspension of 1,1-dimethylethyl 4-(carboxymethyl)phenylpropanoate (5.3 g, 20.0 mmol) and potassium carbonate (2.8 g, 20.0 mmol) in DMF (30 ml) was added benzyl bromide (3.42 g, 20 mmol). After stirring for 24 h, the solvent was removed in vacuo and the residue was extracted with hexanes (100 ml). Filtration and removal of the solvent under reduced pressure gave 1,1-dimethylethyl 3-[4-(2-benzyloxy-2-oxo-ethyl)phenyl]propanoate (6.5 g, 18.4 mmol) as a colorless liquid in 92% yield.

EXAMPLE o a Preparation of 3-[4-(2-benzyloxy-2-oxoethyl)phenyllpropanoic acid 0 00 S, 1,1-dimethylethyl 3-[4-(2-benzyloxy-2-oxoethyl) phenyl]propanoate (3.0 g, 8.5 mmol) was stirred in formic acid (10 ml) for 8 hours. Removal of the solvent in vacuo Sprovided a colorless oil that was shaken vigorously with a 00 25 mixture of ethyl acetate and hexanes 20 ml) until it solidified. The mixture was cooled in ice water and the product, collected by suction filtration to give 3-[4-(2-benzyloxy-2-oxoethyl)phenyl]propanoic acid, as a white solid (2.2 g, 7.4 mmol) in 87% yield, mp. 62-64.5 0

C.

a 0 a d 58 EXAMPLE 26 Preparation of N-Hydroxysuccinyl 3-(4-carboxymethyl)phenyllpropanoate 3-[4-(2-benzyloxy-2-oxoethyl)phenyi]propanoic acid (1.10 g; 3.68 mmol) N-hydroxy succinimide (0.51 g; 4.43 mmol); and 3.86 ml of 1M dicyclohexylcarbodimide in CH2 C 2 were dissolved in CH2C1 2 (50 ml). The mixture was stirred at room temperature for 22 hours.

ml of acetic acid was added and the mixture stirred for 3 hours after which the dicyclohexylurea was removed by filtration. The solvent was removed in vacuo and the residue was redissolved in DMF (40 After standing approx. 30 min. additional dicyclohexylurea was removed by filtration. The volume was adjusted to 100 ml with S0, additional DMF and the solution was transferred to a pressure bottle. After flushing with N 2 10% Pd/C (1.0 g) was added and the bottle was placed on a Parr hydrogenation 20 apparatus. After flushing the system 3 times with 20 psi H, the mixture was shaken under 50 psi H for approx. 1 1/2 hour (or until H 2 up take ceased.) The pressure bottle was then flushed 3x 20 psi N 2 before removing from the parr apparatus. The catalyst was removed by filtration through celite and MgSO 4 The final volume was adjusted to 140 ml with DMF. 35 ml (0.92 mmol) of the solution of N-hydroxysuccinyl 3-(4-carboxymethyl)phenylpropanoate was used per coupling.

30 EXAMPLE 27 Preparation of rac.-2-amino-3-hydroxyphenyl)propanoic acid benzyl ester A 500 ml round bottom flask fitted with a Dean-Stark water separator was charged with a suspension of 10.0 g (0.055 mol) of rac.-3-Hydroxyphenylalanine and 11.5 g (0.066 rrr 59 mol) of p-toluenesulfonic acid monohydrate in 60 ml of benzyl alcohol and 250 ml of toluene. The resulting mixture was heated to reflux for 4 hours as about 2 ml of water was collected in the trap. The mixture was allowed to cool, was diluted with ether and was extracted repeatedly with 1 N hydrochloric acid. The combined extracts were neutralized with excess solid sodium bicarbonate to precipitate 8.72 g of rac.-2-amino-3-(3-hydroxyphenyl)propanoic acid benzyl ester.

EXAMPLE 28 Preparation of rac.-2-acetamido-3-(3-hydroxyphenyl) propanoic acid benzy ester A suspension of 8.72 g (0.032 mol) of rac.-2-amino-3-(3o 0 4 Sr. hydroxyphenyl)propanoic acid benzyl ester in 400 ml of ice cold dichloromethane was treated dropwise with 1.9 ml (0.020 mol) of acetic anhydride. Upon completion of the addition, S 20 a solution of 3.70 g of sodium carbonate in 30 ml of water was added simultaneously with an additional 1.9 ml of acetic anhydride. Afte- 1 hour, the layers were separated and the organic layer was washed with water. The combined aqueous layers were extracted with dichloromethane and the combined extracts were washed with brine and dried over magnesium sulfate. Filtration and concentration afforded a residue which was chromatographed on a Waters Prep 500 liquid chromatograph fitted with two silica gel cartridges, eluting with 20% ethyl acetate-hexane to give 8.49 g of rac.-2-acetamido-3-(3-hydroxyphenyl)propanoic acid benzyl ester as a thick oil.

60 EXAMPLE 29 Preparation of rac.-2-acetamido-3-[3-[(trifluoromethylsulfonyl)oxy]phenyl]propanoic acid benzyl ester A solution of 8.30 g (0.026 mol) of rac.-2-acetamido-3- (3-hydroxyphenyl)propanoic acid benzyl ester and 10.0 g (0.028 mol) of N-phenyl-N-trifluoromethyl- 1,l,1-trifluoromethyl sulfonyl sulfonamide in 110 ml of freshly distilled dichloromethane was cooled in an ice bath and 4.10 ml (0.029 mol) of triethylcmine was added dropwise. The resulting mixture was stirred 1 hour at 0 C and was allowed to warm to room temperature over 2 hours.

The mixture was diluted with 250 ml of ethyl acetate and was washed with successive 50 ml portions of water, 1 N r lium hydroxide, 1 N hydrochloric acid, water, and brine and was o0 dried over magnesium sulfate. Filtration and concentration afforded an oil which was purified by preparative chromatography on a Waters Prep 500 liquid chromatograph fitted with two Prep-Pak silica gel cartridges, eluting with 40% ethyl acetate-hexane to give 10.02 g of rac.-2-acetamido-3-[3-[(trifluoromethylsulfonyl)oxy]phenyl] propanoic acid benzyl ester.

0" 25 EXAMPLE 0*0 Preparation of rac.-2-acetamido-3-f3-(2-propenyl)phenyll propanoic acid benzyl ester 30 Argon was passed through a solution of 9.89 g (0.022 mol) of rac.-2-acetamido-3-[3-(3-trifluoromethylsulfonyl) oxyjphenyl]propanoic acid benzyl ester, 2.8 g (0.066 mol) of lithium chloride, and 7.00 ml (0.0225 mol) of allyltributyltin in 50 ml of dimethylformamide for ten minutes and 0.20 g (0.00028 mol) of bis(triphenylphosphine)palladium dichloride was added. The bath temperature was raised to 95-100 0 C for 2 hours at which time a black *i 9 V.1 4i 0 *4 61 precipitate was observed to form. The mixture was cooled, diluted with 250 ml of ether, was washed with 3 x 50 ml of water and 1 x 50 ml of brine and was dried over magnesium sulfate. Filtration and concentration afforded an oil which was purified by preparative chromatography on a Waters Prep 500 liquid chromatograph fitted with two silica gel cartridges and eluting with 33% ethyl acetate-hexane to give 6.70 g of rac.-2-acetamido-3-[3-(2-propenyl)phenyl] propanoic acid benzyl ester as a white solid, mp 55-56.5 0

C.

A portion was recrystallized from ether-hexane resulting in a mp 57-58 0

C.

EXAMPLE 31 Preparation of rac.-2-acetamido-3-[3-(carboxymethyl) phenyl]propanoic acid benzyl ester Solutions of 6.25 g (0.0185 mol) of rac.-2-acetamido-3- [3-(2-propenyl)phenyl]propanoic acid benzyl ester in 120 ml each of acetonitrile and carbon tetrachloride and 11.9 g (0.0555 mol) of sodium metaperiodate in 240 ml of water were combined and stirred mechanically as 0.25 g (0.0013 mol) of ruthenium chloride hydrate was added to the mixture. The mixture darkened immediately and after 1 hour, was diluted 25 with 300 ml of dichloromethane. The layers were separated and the organic layer was washed with 100 ml of water. The combined aqueous layers were extracted with 200 ml of ether and the combined extracts were dried over magnesium sulfate, filtered and concentrated.

The residue was dissolved in 180 ml of tert-butyl alcohol and 60 ml of 2-methyl-2-butene and a solution of 19.0 g (0.21 mol) of sodium chlorite and 19.0 g (0.137 mol) of monobasic sodium phosphate in 130 ml of water was added all at once. The mixture was stirred at 0 0 C for 2 hours, was diluted with ether and the layers were separated. The organic layer was washed with 10% sodium thiosulfate and i ,1 :i i "i i i ii i:l i,

B

I

rr-- 544

C',

-r c 62 brine and was dried over magnesium sulfate. Filtration and evaporation gave a oily residue which was chromatographed over 200 g of silica gel eluting with 60:40:1 ethyl acetate-hexane-acetic acid to give 4.64 g of a white solid, mp 129-139 0 C. Recrystallization from dichloromethanehexane gave 4.14 g of rac.-2-acetamido-3-[3- (carboxymethyl)phenyl]propanoic acid benzyl ester, mp 130-131 0

C.

EXAMPLE 32 Preparation of rac.-2-acetamido-3-[3-[2-(l,l-dimethylethoxy)-2-oxoethyl]benzene]propanoic acid benzyl ester A suspension of 4.00 g (0.0113 mol) of rac.-2-acetamido -3-[3-(carboxymethyl)benzene]propanoic acid benzyl ester in 0 40 ml of toluene and 15 ml of dimethylformamide di-tertbutyl acetal was heated to a bath temperature of 55 0

C.

S° After 30 min, a clear solution formed and after 3 hours, the 20 mixture was cooled, diluted with 50 ml of ether and washed with 3 x 25 ml of water, 1 x 25 ml of brine and was dried over magnesium sulfate. Filtration and concentration gave a white solid which was recrystallized from ether-hexane to afford 3.90 g of rac.-2-acetamido-3-[3-[2-(1,1- 25 dimethylethoxy)-2-oxoethyl]benzene]propanoic acid benzyl ester, mp 80-83 0

C.

EXAMPLE 33 Preparation of rac.-2-acetamido-3-[3-r2-(1,1-dimethylethoxy)-2-oxoethyl]benzene propanoic acid A suspension of 3.30 g (0.00802 mol) of rac.-2-acetamido -3-[3-[2-(1,1-dimethyeththoxy)-2-oxoethylpropanoic acid benzyl ester in 50 ml of ethanol was hydrogenated over 200 mg of 10% palladium on carbon for 3 hours at room temperature under 1 atmosphere of hydrogen. Hydrogen uptake i -63 amounted to 220 ml at which time the mixture was filtered and concentrated to afford 2.54 g of a white solid, mp 167-170 0 C. Recrystallization from ethanol-hexane afforded 1.77 g of rac.-2-acetamido-3-[3-[2-(l,1dimethylethoxy)-2-oxoethyl]benzene propanoic acid, mp 169-172 0

C.

EXAMPLE 34 Preparation of 1,1-dimethylethyl 4-[[2-(1,1-dimetethylethl)- A mixture of Pd(OAc) (90 mg, 0.42 mmol) and 2 1,1'-bis(diphenylphosphino)ferrocene (300 mg, 0.56 mmol) in DMF (40 ml) was heated at 90 0 C for 15 minutes while argon was being passed through the mixture. After cooling the burgundy solution to ambient temperature, 5-[(4-bromophenyl) So methyl]-2-(l,1-dimethylethyl)-2H-tetrazole (4.0 g, 14 mmol), t-butyl acrylate (3.1 g, 23 mmol) and triethylamine (2.8 ml, 00 09 S 20 20 mmol) were added to the mixture. The reaction was heated S, at 90 0 C overnight and then concentrated. After filtering the dark mixture through a short column of silica gel using a mixture of hexanes and ethyl acetate the fractions containing product were pooled and concentrated. Final 25 purification was achieved by chromatography on a Waters Prep 500 liquid chromatograph fitted with two silica gel cartridges eluting with a mixture of hexanes and ethyl acetate to give pure 1,1-dimethylethyl 4-[[2-(1,1-dimethyethylethy)-2H-tetrazol-5-yl]methyl]benzene propenoate (3.9 g, 11.3 mmol) in 81% yield. The analytical sample was recrystallized from ethyl acetate and hexanes, mp. 105-106 0

C.

L m 64 EXAMPLE Preparation of 1,l-dimethylethrl 4-fr2-(l1j-dimethlethl)- 2H-tetrazol--vxlmethyllbenzene pro panoate 1,1-dimethylethyl 4-[[2-(]1,l-dimethiylethyl)propanoate (3.4 g, 9.9 mmol) in ethanol (30 ml) containing 10% Pd/C (200 mg) was stirred under a blanket of hydrogen (1 atm). After hydrogen uptake ceased, the mixture was filtered through a pad of Celite followed by additional ethanol (30 ml). The filtrate was concentrated and then purified by chromatography on a Waters Prep 500 liquid chromatograph fitted with two silica gel cartridges eluting with a mixture of hexanes and ethyl 1acetate followed by recrystallization from hexanes to give pure 1,1-dimethylethyl 4-[[2-(1,1-dimethylethyl)- 0 2H-tetrazol-5-yljmethyl~benzenepropanoate (3.0 g, 8.6 mmol) in 87% yield, mp. 54-55 0

C.

EXAMPLE 36 Preparation of 4-fr2--(1,1-dimethylethyl)-2H-tetrazol-5-ylI methyllbenzene propanoic acid 1,1-dimethylethyl 4-f E2-(1,1-dimethylethyl)-2H-tetrazol- (3.0 g, 8.7 mmol) was dissolved in trifluoroacetic acid (33 ml) and stirred at room temperature under argon for 12 hours. The mixture was concentrated and then dissolved in water (50 ml). After adding enough sat. NaHCO 3 to raise the pH to 8, the solution was washed with diethyl ether (2 x 25 ml). The aqueous layer was acidified to pH 3 with 1N HCl and the product collected by suction filtration. Recrystallization from hexanes gave 4-112-(l,1-dimethylethyl)-2H-tetrazol-5-yljmethyllbenzene-propanoic acid (1.5 g, 5.7 mmfol) in 66% yield. mp. 139-141 0

C.

65 EXAMPLE 37 Preparation of 5-(4-bromophenyl)-2H-tetrazole 4-Bromobenzonitrile (20 g, 110 mmol), sodium azide (7.9 g, 121 mmol) and NH Cl (6.5 g, 121 mmol) in DMF (340 ml) were heated at 90 0 C under argon. After 2 days the reaction mixture was concentrated and then diluted with water (300 ml). After adding enough 1M NaOH to render the mixture basic it was washed with ether (4 x 25 ml). The aqueous portion was acidified to pH 3 with IN HC1, the precipitated product was collected by suction filtration and then washed with water. The crude product was recrystallized from ethanol to give crystalline 5-(4-bromophenyl)-2H-tetrazole (19.2 g, 83.6 mmol) in 76% S'o yield, mp. 271-273 0

C.

o oo EXAMPLE 38 S 20 Preparation of 5-(4-bromophenyl)-2-(1,1-dimethylethyl)- 2H-tetrazole 5-(4-Bromophenyl)-2H-tetrazole (19.0 g, 81.9 mmol), t-butanol (12.1 g, 164 mmol), trifluoroacetic acid (80 ml) S 25 and concentrated sulfuric acid (4.6 g, 41 mmol) were stirred at ambient temperature for 24 hours. The mixture was concentrated and dissolved in ethyl acetate (200 ml). After washing with water (3 x 25 ml), 1M NaOH (3 x 25 ml) and brine (25 ml) the organic layer was dried (Na 2

SO

4 filtered and concentrated. The oil was purified by chromatography on a Waters Prep 500 liquid chromatogcaph fitted with two silica gel cartridges eluting with a mixture of hexanes and ethyl acetate to provide 5-(4-bromophenyl)-2-(1,1-dimethylethyl)-2H-tetrazole (17.1 g, 58.1 mmol) as a yellow oil in 71% yield.

66 EXAM~PLE 39 Preparation of 1,1-dimethylethyl 4-[2-(1,l-dimethylethyl)lbenzenepropenoate A solution of 5-(4--bromopbhenyl)-2-(1,1-dimethylethyl)- 2H-tetrazole (3.0 g, 10.2 mmol), t-butyl acrylate (2.9 ml, 20.4 mmol), and triethylamine (7.9 ml, 59 mmol) in DMF (93 ml) was deoxygenated with argon, whereupon bis(triphenylphiosphine)palladium dichloride (0.5 g, 0.8 mmol) was added. After heating at 75 0 C for 12 hours the mixture was concentrated under reduced pressure and the brown residue was filtered through a short column of silica gel eluting with a mixture of hexanes and ethyl acetate The eluent was concentrated and then purified by chromatography on a Waters Prep 500 liquid chromatograph fitted with two silica gel cartridges eluting with a mixture of hexanes and ethyl acetate The fractions a containing product were pooled, concentrated and the residue was recrystallized from hexanes to give 1,1-dimethylethyl 8 4-[2-(1,1-dimethylethyl)-2H-tetrazol-5-yllbenzene propenoate (1.6 g, 5.6 mmol) in 55% yield, mp. 118.5-120 0

C.

EXAMPLE Preparation of l-dimethylethoxy)-3-oxgoprpyl) phenyll-2-(1,1--dimethylethyl)-2H-tetrazole A suspension of 1,1-dimethylethyl 4-r2-(1,l-dimethylethyl)-2H-tetrazol-5-yljbenzene propenoate (1.6 g, 5.6 mmol) and 100 mg of 10% Pd/C in ethanol (17 ml) was stirred under a blanket of hydrogen (1 atm). After hydrogen uptake ceased, the mixture was filtered through a pad of Celite washing with ethanol (30 ml). The filtrate was concentrated to give 5-[4-(3-(l,1-dimethylethoxy)-3oxopropyl)phenylj-2-(1,1-dimethylethyl)-21-tetrazole (1.2 g, 4.4 mmol) in 78% yield.

-67- EXAMPLE 41 Preparation of 4-F2-(1,1-dimethylethyl)-2H-tetrazol-5-yl1benzenepropanoic acid 5-[4-(3-(l,l-dimethylethoxy)-3-oxopropyl)phenyl]-2- (l,1-dimethylethyl)-2H-tetrazole (1.0 g, 3.7 mmol) was dissolved in trifluoroacetic acid (14 ml) and stirred at room temperature under argon for 12 hours. The mixture was concentrated and then dissolved in water (35 ml). After adding enough sat. NaHCO 3 to raise the pH to 8, the solution was washed with diethyl ether (2 x 10 ml). The aqueous layer was acidified to pH 3 with lN HC1 and the product was collected by suction filtration to give 4-[2-(1,1-dimethylethyl)-2H-tetrazole-5-yl]benzenepropanoic acid (0.7 g, 3.1 mmol) in 84% yield, mp. 88-90 0

C

0a EXAMPLE 42 0 q S' 20 Preparati of Ac-(D,L)Phe(3-COOH)-Met-Gly-Trp-Met-Asp- Phe-NH 2 6 g of Boc-Phe-PAM resin (substitution 0.36 mmol/g) was suspended and shaken in TFA/CH 2 Cl 2 (1:1 by volume, 3 x F 2. 50 ml) 10 min. each time at room temperature to remove the Boc-group. The product was isolated by filtration and washed (3 x 50 ml each) with CH 2 C1 2 8% DIEA in CH2C1 and CH2Cl to give the free base of Phe-PAM-resin. This was subjected to sequential solid phase synthesis using the Fmoc-protocol. All couplings were performed using the DCC/HOBt procedure. At step 7 the Fmoc-amino acids, DCC, and HOBt were added with the corresponding reaction times as follows: Fmoc-Asp(OtBu)-OH (2.46 g, 6 mmol), DCC (1.24 g, 6 mmol) and HOBt (1.2 g, 9 mmol) were dissolved in 50 ml of 1:1 by volume DMF/CH C1 2 and allowed to couple for 60 min at room temperature. Fmoc-Met-OH (2.2 g, 6 mn.ol), DCC (1.24 g, 68 6 mmol) and HOBt (1.2 g, 9 mmol) were dissolved in 50 ml of 1:1 by volume DMF/CH 2 Cl 2 and allowed to couple for min at room temperature. Fmoc-Trp-OH (2.6 g, 6 mmol), DCC (1.24 g, 6 mmol) and HOBt (1.2 g, 9 mmol) were dissolved in 50 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for 60 min at room temperature. Fmoc-Gly-OH (1.8 g, 6 mmol), DCC (1.24 g, 6 mmol) and HOBt (1.2 g, 9 mmol) were dissolved in 50 ml of 1:1 by volume DMF/CH 2 C1 2 and allowed to couple for 60 min. Fmoc-Met-OH (2.2 g, 6 mmol), DCC (1.24 g, 6 mmol) and HOBt (1.2 g, 9 mmol) were dissolved in 50 ml of 1:1 by volume DMF/CH Cl 2 and allowed to 2 2 couple for 60 min at room temperature. At this point the peptide-resin was dried under high vacuum to provide 7.98 g of Fmoc-Met-Gly-Trp-Met-Asp(OtBu)-Phe-PAM resin.

A portion (1.5 g, 0.4 mmol) of Fmoc-Met-Gly-Trp-Met-Asp- O. (OtBu)-Phe-PAM resin was deprotected with 20% piperidine/DMF o (step 1-6) using the Fmoc protocol and coupled to the compound of Example 17(a), rac.-2-acetamido-3-[3-[1,1dimethyl ethoxy)carbonyl]phenyl]propanoic acid [Ac-(D,L)Phe (3-COOtBu)] (400 mg, 1.5 mmol), using DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) which were dissolved in 50 ml of DMF/CH Cl1 by volume and allowed to react for min at room temberature, then washed (step 8-16), and dried to yield Ac-(D,L)Phe(3-COOtBu)-Met-Gly-Trp-Met-Asp(OtBu)- Phe-PAM-resin. This peptidyl-resin was suspended and shaken in 50% TFA/CH Cl with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the OtBu groups. The peptidyl-PAM resin was then isolated by filtration, washed (3 x 50 ml each) with CH2 Cl, DMF and methanol and placed in a pressure bottle, suspended in 100 ml methanol, saturated with NH 3 at -20 0 C and sealed. The suspension was stirred at room temperature for 3 days. After venting the excess NH 3 the PAM-resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness to give 1.00 g of crude peptide.

J I la I I I\ i I ~L 69 100 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C18 column. The peptide was eluted with a linear gradient (4h) of 5% to 0.022% TFA/CH3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 25 mg of Ac-(D,L)Phe(3-COOH)-Met-Gly-Trp-Met- Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.00(1); Gly, 1.00(1), Met, 1.95(2); Phe, 1.01(1); Trp, 0.80(1); (D,L)Phe(3-COOH), n.d. Empirical Formula: C H N 90 2S. M.W. 1018.18.

EXAMPLE 43 Preparation of Ac-(D,L)Phe(4-COOH)-Met-Gly-Trp-Met-Asp- _Phe-NH 2 o 1.76 g (0.47 mmol) of Fmoc-Met-Gly-Trp-Met-Asp(OtBu)-Phe- PAM-resin obtained from Example 42 was deprotected with 6 a 9 piperidine/DMF (step 1-6) using the Fmoc protocol and coupled to the compound of Example 17, rac.-2-acetamido-3- [4-[(1,1-dimethylethoxy)carbonyl]phenyl]propanoic acid [Ac-(D,L)Phe(4-COOtBu)] (700 mg, 1.5 mmol) using DCC S(310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) which were 9 25 dissolved in 50 ml of DMF/CH 2 Cl 2 by volume and allowed to react for 60 min at room temperature, then washed (step 8-16) and dried to yield Ac-(D,L)Phe(4-COOtBu)-Met-Gly- Trp-Met-Asp(OtBu)-Phe-PAM-resin. This peptidyl-resin was suspended and shaken in 50% TFA/CH2Cl 2 with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the OtBu groups. The peptidyl-resin was then isolated by filtration, washed (3 x 50 ml each) with CH Cl 2 DMF and methanol and placed in a pressure bottle suspended in 100 ml of methanol, saturated with NH 3 at -20 0 C and sealed. The suspension was stirred at room temperature for 3 days.

L_ _ri 70 After venting the excess NH 3 the PAM-resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness to yield 541 mg of crude peptide.

80 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C18 column. The peptide was eluted with a linear gradient (4h) of 5% to 0.022% TFA/CH3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 23 mg (33 of Ac-(D,L)Phe(4-COOH)-Met-Gly-Trp-Met- Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.00(1); Gly, 0.98(1); Met, 2.00(2); Phe, 1.00(1); Trp, 0.76(1); (D,L)Phe(4-COOH), n.d. Empirical Formula: C48H59 N902S2 M.W. 1018.18.

00 0o EXAMPLE 44 0 0

OQ

0 Preparation of Ac-(D)Phe(4-COOH)-Met-Gly-Trp-Met-Asp- 0O '0 0 o 20 Phe-NH o o 4 mg of Ac-(D,L)Phe-(4-COOH)-Met-Gly-Trp-Met-Asp-Phe-

NH

2 were dissolved in 0.5 ml of 0.2N NH OH and applied on o to (1.25 x 30) cm micro Bondapack C column from E.S.

18 co' 25 Industries. The column was previously equilibrated with CH CN/O.01M NH OAc and eluted with a linear gradient of 00o 10-40% CH 3 CN in 0.01M NH4OAc for 120 min at 5 ml/min.

The 280 nm absorption of the column effluent was monitored.

Two peaks were detected eluting at 78 min at 84 min.

ooo 30 Fractions containing these peaks were pooled and lyophilized 0 to 1.5 mg of white powder. Analysis by Glass Capillary Gas Chromatography shows that the compound at 78 min retention time contains the (D)Phe(4-COOH) enantiomer. The purity of this compound was also determined by analytical HPLC, amino acid analysis, UV and IR.

i _1 i 71 EXAMPLE Preparation of Ac-Phe(4-COOH)-Met-Gly-Trp-Met-Asp- Phe-NH 2 From Example 44, the compound eluting at 84 min retention time was analyzed by Glass Capillary Gas Chromatography and showed to contain the (L)Phe(4-COOH) enantiomer. The purity of this compound was also determined by analytical HPLC, amino acid analysis, UV and IR.

EXAMPLE 46 Preparation of Ac-(D,L)Phe(4-CH COOH)-Met-Gly-Trp-Met- Asp-Phe-NH2 1.00 g (0.27 mmol) of Fmoc-Met-Gly-Trp-Met-Asp(OtBu)-Phe- .o PAM-resin obtained from Example 42 was deprotected with piperidine/DMF (step 1-6) using the Fmoc protocol and 0 0 20 coupled to the compound of Example 17(b,) rac.-2-acetamido o 0 0 S -3-[4-[l,l-dimethylethoxy)-2-oxoethyl]carbonyl]phenyl] p opanoic acid [Ac-(D,L)Phe(4-CH2COOtBu)] (280 mg, 0.8 mmol) using the DCC (165 mg, 0.8 mmol) and HOBt (200 mg, 1.5 mmol) which were dissolved in 40 ml DMF/CH 2 Cl1 (1:1) a 2 2 Oo 25 by volume and allowed to react for 60 min at room temperature, then washed (step 8-16) and dried to yield o Ac-(D,L)Phe(4-CH COOtBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe- 0.

PAM-resin. This peptidyl-resin was suspended and shaken in TFA/CH2C12 with 1% EDT (2 x 50 ml) 10 min each time 30 at room temperature to remove the OtBu groups. The S. o peptidyl-resin was then isolated by filtration, washed (3 x 50 ml each) with CH2 Cl, DMF and methanol, and placed in a pressure bottle, suspended in 100 ml of methanol, saturated with NH at -20 0 C and sealed. The suspension was stirred at room temperature for 3 days.

After venting the excess NH 3 the PAM-resin was filtered off and washed with methanol. The combined filtrates were 72 evaporated to dryness to yield 392 mg of crude peptide.

mg of crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C18 column. The peptide was eluted with a linear gradient (4h) of 5% to 65% 0.022%

TFA/CH

3 CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 13 mg (24 of Ac-(D,L)Phe(4-CH 2 COOH)-Met-Gly-Trp- Met-Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.02(1); Gly, 1.00(1); Met, 1.96(2); Phe, 1.00(1); Trp, 0.75(1); Phe(4-CH2COOH), 1.00(1). Empirical Formula: C49H6N 0 12S M.W. 1032.20.

EXAMPLE 47 Preparation of Ac-(D)Phe(4-CH COOH)-Met-Gly-Trp- -2 Met-Asp-Phe-NH 2 mg of Ac-(D,L)Phe(4-CH2COOH)-Met-Gly-Trp-Met-Asp- Phe-NH were dissolved in 0.5 ml of 0.2N NH4OH and applied to (2.3 x 30) cm micro Bondapack C18 column from E.S. Industries. The column was previously equilibrated with 2% CH3CN/0.01M NH 4 OAc and eluted with a linear gradient of 2-20% CH CN in 0.01M NH OAc over 5 min at S 25 8 ml/min, then held at 20% CH3CN for 120 min. The 280 nm absorption of the column effluent was monitored. Two peaks were detected eluting at 40 min and 46 min. Fractions containing these peaks were pooled and lyophilized to 0.8 mg of white powder. Analysis by Glass Capillary Gas S 30 Chromatography shows that the compound eluting at 40 min retention time contains the (D)Phe(4 -CH 2 COOH) enantiomer.

The purity of this compound was als- confirmed by analytical HPLC, amino acid analysis, UV and IR.

c i r 73 EXAMPLE 48 Preparation of Ac-Phe(4-CH2COOH)-Met-Gly-Trp-Met-Asp- Phe-NH 2 From Example 47, the compound eluting at 46 min retention time was analyzed by Glass Capillary Gas Chromatography and showed to contain the (L)Phe(4-CH 2

COOH)

enantiomer. The purity of this compound was also determined by analytical HPLC, amino acid analysis, UV and IR.

EXAMPLE 49 0040 9 0 00 0 0 0 o 4 00 Preparation of Ac-(D)Phe(4-CH2COOC2H5)-Met-Gly-Trp- Met-Asp(COOC2H )-Phe-NH 15 mg of Ac-(D,L)Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp- Phe-NH 2 (0.015 mmol) were dissolved in 4 ml of 2:1 by volume ethanol/DMF, HOBt (6 mg, 0.04 mmol) was added and the 20 mixture was cooled to 0°C, DCC (9 mg, 0.04 mmol) was then added and the reaction mixture was stirred for 1 hr at 0 C and allowed to stand at 5 0 C for 24 hours and at room temperature for 15 hours. The reaction mixture was then evaporated in vacuo. The residue was taken off in 1 ml of DMF and applied into a (2.3 x 30) cm micro Bondapack C 18 column. The column was previously equilibrated with CH3CN/0.022% TFA and eluted with a linear gradient of 5-65% CH3CN in 0.022% TFA over 240 min at 8 ml/min. The 280 nm absorption of the column effluent was monitored. Two peaks were detected eluting at 168 min and 171 min.

Fractions containing these peaks were pooled and lyophilized to yield 4 mg of white powder. Analysis by MS, NMR and Glass Capillary Gas Chromatography shows that the compound eluting at 168 min retention time is: Ac-(D)Phe(4-CH2COOC2H5)-Met-Gly-Trp-Met-Asp(COOC2H5 -Phe-NH 2 Empirical Formula: C53H69N 012S2' M.W. 1088.31.

IL ii 74 EXAMPLE Preparation of Ac-Phe(4-CH 2COOC 2H Met-Asp-(COOC 2 H11 5 )-Phe-NH 2 From Example 49. the compound eluting at 171 min retention time was analyzed by MS, NMR and Glass Capillary Gas Chromatography and showed to be Ac-he(-CH2 CC 2 H 5 )-Met-Gly-Trp-Met-Asp(COOC 2

H

5 Phe.-NH 2 EXAMPLE 51 Preparation of Ac-Phe(4-CH 2CH 2COOH)-Met-Gly- TrD-Met-Asv-Phe-NH_ o 4 44 4 04 44 44 4 4 £4 I 4 4 44 4 4 I 44 I C I 4 41 1.76 g (0.47 mmol) of Fmoc-Met-Gly-Trp--Met-Asp(OtBu)-Phe- PAM-resin obtained from Example 42 was deprotected with piperidine/DMF (step 1-6) using the Emoc protocol and 20 coupled to the compound of Example 20 dimethylethoxy)carbonyl~amino]-3-[4-[3- 1-dimethylethoxy) 3-oxu-1-propenyllphenyljpropaioic acid [Boc-Phe(4-CH 2CH 2COOtBu)] (550 mg, 1.5 mmol) using DCC (310 mng, 1.5 mmol) and HOBt (270 mg, 2 mmol) which were 25 dissolved in 50 ml of DMF/CH 2Cl 11 yvlm n allowed to react for 60 min at room temperature then washed (step 8-16) and dried to yield Boc-Phe(4-CH 2 CH 2 COOtBu)- Met-Gly-Trp-Met-Asp(OtBu)-Phe-PAM-resin. This peptidyl-resin was suspended and shaken in TFA/CH 2 Cl 2 with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the Boc and OtBu groups. The peptidyl-resin was then isolated by filtration, washed (3 x 50 ml each) with CH 2Cl 2'DMF and methanol, and then acetylated using 3 equivalents of acetic acid and BOP reagent in the presence of 1.5% DIEA according to the same protocol as used for the Boc-amino acid residues. The acetylated peptidyl-resin was then placed in a pressure _I L 1_I/ Il _ii 75 bottle, suspended in 75 ml of methanol saturated with NH 3 at -20 0 C and sealed. The suspension was stirred at room temperature for 3 days. After venting the excess NH 3 the PAM resin was filtered off and washed with methanol and acetic acid. The combined filtrates were evaporated and the residue lyophilized to yield 831 mg of crude peptide.

100 mg crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C18 column. The peptide was eluted with a linear gradient (4h) of 5% to 65% 0.022%

TFA/CH

3 CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 17mg of Ac-Phe(4-CH CH COOH)-Met-Gly-Trp- Met-Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.00(1); Gly, 1.01(1); Met, 1.95(2); Phe, 1.00(1); Phe(CH 2

CH

2 COOH), 0.97(1); Trp, 0.70(1).

Empirical Formula: C H63N 012S 2 M.W. 1046.22.

50 63 9 12 2 00 0 S EXAMPLE 52 a o Preparation of Ac-(D,L)Phe(4-CF COOH)-Met-Gly- Trp-Met-Asp-Phe-NH 2 1.00 g (0.27 mmol) of Fmoc-Met-Gly-Trp-Met-Asp(OtBu)-Pheo.0 25 PAM-resin obtained from Example 42 was deprotected with piperidine/DMF (step 1-6) using the Fmoc protocol and 0:0" coupled to the compound of Example 17(d), rac.-2-acetamido -3-[4-[1,1-difluoro[2-(1,1-dimethylethoxy)-2-oxoethyl]phenyl] propanoic acid [Ac-(D,L)Phe(4-CF2COOtBu)] (310 mg, 30 0.8 mmol) using the DCC (165 mg, 0.8 mmol) and HOBt (200 mg, o 1.5 mmol) which were dissolved in 40 ml DMF/CH Cl (1:1) by volume and allowed to react for 60 min at room temperature then washed (step 8-16) and dried to yield Ac-(D.L)Phe(4-CF 2 COOtBu)-Met-Gly-Trp-Met-Asp(OtBu)-Phe-PAMresin. This peptidyl-resin was suspended and shaken in TFA/CH2Cl 2 with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the OtBu groups. The 76 peptidyl-resin was then isolated by filtration, washed (3 x 50 ml each) with CH 2 C1 2 DMF and methanol and placed in a pressure bottle suspended in 100 ml of methanol, saturated with NH 3 at -20 0 C and sealed. The suspension was stirred at room temperature for 3 days. After venting the excess NH 3 the PAM-resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness to yield 388 mg of crude peptide. 70 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C 18 column. The peptide was eluted with a linear gradient (4h) of 5 to 65% 0.022% TFA/CH 3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 14 mg (27 of Ac-(D,L)Phe(CF 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.00(1); Gly, 1.00(1); Met, 2.04(2); Phe, 1.00(1); Trp, 0.70(1); Phe *(4CF COOH) n.d. Empirical Formula: C49H 59N9 0 12F2S2. M.W. 1068.20.

EXAMPLE 53 Preparation of Ac-(D,L)Phe(4-tetrazole)-Met-Gly-Trp-Met- Asp-Phe-NH 2 S' Boc-Phe (2.6 g, 10 mmol) and HOBt (2.0 g, 15 mmol) were I dissolved in a mixture of 20 ml CH Cl and 20 ml DMF 2 2 chilled to 0°C and with stirring (2.06 g, 10 mmol) DCC was added and the mixture was stirred for 60 minutes at 0 C.

Separately 10 g of benzhydrylamine copolysterene 1% Sdivinylbenzene cross-linked resin (0.56 mmole N/g) was washed with 10% diisopropylethylamine in methylene chloride for 30 min, filtered and washed with methylene chloride dimethylformamide and methylene chloride. The chilled mixture above was added to the resin and stirred for 24 hours at room temperature. The resin was filtered and washed with methylene chloride, dimethylformamide, i 77 isopropanol, methylene chloride, dimethylformamide, isopropanol, methylene chloride and dried under high vacuum. Amino acid analysis showed the resin to contain 0.32 mmoles of phenylalanine per gram of resin. Unreacted amino groups were capped by shaking the resin with 5 ml of acetic anhydride and 5 ml diisopropylethylamine in methylene chloride for 60 minutes. The resin was filtered and washed with methylene chloride, isopropanol, dimethylformamide and methylene chloride. 1.5 g (0.48 mmol) Boc-Phe-BHA resin was subjected to sequential solid phase synthesis using the Boc protocol. All couplings were performed using the DCC/HOBt procedure. At step 16 the Boc-amino acid, DCC and HOBt were added with the corresponding reaction times as follows: Boc-Asp(OBzl)-OH (485 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH 2

C

2 l and allowed to couple for 60 min at 4° room temperature. Boc-Met-OH (380 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH Cl 2 and allowed to 20 couple for 60 min at room temperature. Boc-Trp(For)-OH (500 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of DMF, and allowed to couple for 60 min at room temperature. Boc-Gly-OH (270 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) 25 were dissolved in 20 ml of 1:1 by volume DMF/CH2C12 and o0 2 2 2 allowed to couple for 60 min at room temperature.

.00. Boc-Met-OH (380 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for 60 min at room temperature. The compound of Example 17(c), rac.-2acetamido-3-[4-[5-(2-(l,1-dimethylethyl)-2H-tetrazoyl]] phenyl]propanoic acid [Ac-(D,L)Phe(4-tetrazole-tBu)] (500 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2 Cl, and allowed to couple for 60 min at room temperature. At this point the peptidyl-resin was dried

L

-I-

-rr~- 78 under high vacuum to provide 2.25 g of Ac-(D,L)Phe(4tetrazole-tBu)-Met-Gly-Trp(For)-Met-Asp(OBzl)-Phe-BHA-resin.

2.25 g of the resin was cleaved by treatment with 5 ml of HF containing 2.0 ml of anisole, 1.0 ml of EDT and 15 ml of dimethylsulfide for 1 hour at 0 C. After evaporation to a low volume, fresh anhydrous HF (20 ml) was distilled into the reaction vessel for a second treatment for 2h at 0°C.

After thorough evaporation, the resin was washed with 2 volumes of ethylacetate then triturated with 4 x 15 ml of acetic acid, filtered and lyophilized to yield 415 mg of crude peptide.

100 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C 18 column. The peptide was eluted with a linear gradient (4h) of 5 to 0.022% TFA/CH3CN of a flow rate of 8 ml/min, detection at S. 280 nm. The main peak was collected and lyophilized to S°'o yield 8 mg of Ac-(D,L)Phe(4-tetrazole)-Met-Gly-Trp- S, 20 Met-Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 0.98(1); Gly, 1.00(1); Met, 1.92(2); Phe, 1.00(1); (D,L)Phe(4-tetrazole), 0.96(1); Trp, n.d.

Empirical Formula: C48H59 N 30 S 2 M.W. 1042.19.

EXAMPLE 54 Preparation of Ac-(D)Phe(4-tetrazole)-Met-Gly-Trp-Met- Asp-Phe-NH 2 S2 mg of Ac-(D,L)Phe(4-tetrazole)-Met-Gly-Trp-Met-Asp- Phe-NH 2 from example 53 were dissolved in 0.5 ml of 0.2N NH4OH and applied to (2.3 x 30) cm micro Bondapack C 18 column from E.S. Industries. The column was previously equilibrated with 1% CH3CN/0.01M NH4OAc and eluted with a step gradient of 1-20% CH3CN/0.O1M NH4 OAc over 5 min at 8 ml/min, then 20-30% CH3CN/O.01M NH4OAc over 120 min c F~ iii-i 79 at 8 ml/min. The 280 nm absorption of the column effluent was monitored. Two peaks were detected eluting at 35 min and 39 min. Fractions containing these peaks were pooled and lyophilized to 1 mg of white powder. Analysis by Glass Capillary Gas Chromatography shows that the compound eluting at 35 min retention time contains the (D)Phe(4-tetrazole) enantiomer. The purity of this compound was also confirmed by analytical HPLC, amino acid analysis, UV and IR.

EXAMPLE Preparation of Ac-Phe(4-tetrazole)-Met-Gly-Trp-Met- Asp-Phe-NH 2 From Example 54, the compound eluting at 39 min retention time was analyzed by Glass Capillary Gas c° Chromatography and shown to contain the (L)Phe(4-tetrazole) o enantiomer. The purity of this compound was also determined by analytical HPLC, amino acid analysis, UV and IR.

oa oo 0 oo EXAMPLE 56 Preparation of Ac-(D,L)Phe(4-CH COOH)-Lys-Gly-Trp-Met-Asp-N- 2 d methyl-Phe-NH o o 2 Boc-N-methyl-Phe (5 g, 17.8 mmol) and HOBt (34 g, mmol) were dissolved in a mixture of 20 ml CH Cl and 0000 2 2 ml DMF chilled to OOC and with stirring (4.12 g, 20 mmol) DCC was added and the mixture was stirred for 60 minutes at o 30 0 0 C. Separately 10 g of benzhydrylamine copolysterene 1% o0, divinylbenzene cross-linked resin (0.39 mmol N/g) was washed with 10% diisopropylethylamine in methylene chloride for min, filtered and washed with methylene chloride dimethylformamide and methylene chloride. The chilled mixture above was added to the resin and stirred for 24 hours at room temperature. The resin was filtered and washed with methylene chloride, dimethylformamide, c I 3C i'i 80 isopropanol, methylene chloride, and dried under high vacuum. Amino acid analysis showed the resin to contain 0.30 mmoles of N-methylphenylalanine per gram of resin.

Unreacted amino groups were capped by shaking the resin with 5 ml of acetic anhydride and 5 ml diisopropylethylamine in methylene chloride for 60 min. The resin was filtered and washed with methylene chloride, isopropanol, dimethylformamide and methylene chloride.

4 g (1.2 mmol) Boc-N-methyl-Phe-BHA resin was suspended and shaken in TFA/CH Cl2 by volume (3 x 40 ml) min, each time at room temperature to remove the Boc group.

The product was isolated by filtration and washed (3 x 50 ml each) with CH2C1 2 8% DIEA in CH Cl 2 and CH2C12 to give the free base of N-methyl-Phe-BHA resin. This was subjected to sequential solid phase peptide synthesis using S the DCC/HOBt procedure. At step 7 the Fmoc amino acid, DCC and HOBt were added with the corresponding reaction times as follows: Fmoc-Asp(OtBu)-OH (1.64 g, 4 mmol), DCC (825 mg, 20 4 mmol) and HOBt (810 mg, 6 mmol) were dissolved in 40 ml of .1:1 by volume DMF/CH2C1 2 and allowed to couple for min at room temperature. Fmoc-Met-OH(1.5 g, 4 mmol), DCC (825 mg. (1 mmol) and HOBt (810 mg, 6 mmol) were dissolved in 40 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to 25 couple for 60 min. at room temperature. Fmoc-Trp-OH (1.7 g,4 mmol), DCC (825 mg, 4 mmol) and HOBt (810 mg, 6 mmol) were dissolved in 40 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for 60 min at room temperature. Fmoc-Gly-OH (1.2 g, 4 mmol), DCC (825 mg, 4 mmol) and HOBt (810 mg, 6 mmol) were dissolved in 40 ml of 1:1 by volume DMF/CH2C1, and allowed to couple for 2 2 min at room temperature. Fmoc-Lys(Boc) (1.9 g, 4 mmol), DCC (825 mg, 4 mmol) and HOBt (810 mg, 6 mmol) were dissolved in ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for 60 min. At this point the peptide-resin was dried under high vacuum to provide 5.2 g of Fmoc-Lys(Boc)-Gly-Trp-Met- Asp(OtBu)-N-methyl-Phe-BHA-resin. This peptidyl-resin was c~ i 1 i 81 suspended and shaken in 50% TFA/CH Cl 2 with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the OtBu and the Boc groups. The peptidyl-BHA resin was then neutralized with 8% DIPEA in CH2 Cl, washed (3 x 50 ml each) with CH2C1l DMF and methanol, and isolated by filtration to yield Fmoc-Lys- Gly-Trp-Met-Asp-N-methyl-Phe-BHA resin. The cyclization on the resin between the e-amino group of Lys and the 1-carboxyl group of Asp was achieved using the BOP reagent (2.0 g, 4 mmol) in DMF (50 ml) containing 1.5% DIEA (v/v) for 48h. A negative Kaiser ninhydrin test was observed and the peptide-resin was washed and dried to yield: Fmoc-Lys-Gly-Trp-Met-Asp-N-methyl-Phe-BHA-resin.

1.1 g (0.33 mmol) of Fmoc-Lys-Gly-Trp-Met-Asp-Nmethyl-Phe-BHA-resin was deprotected with 20% piperidine/DMF (step 1-6) using the Fmoc protocol and coupled to the compound of Example 17(b), rac.-2-acetamido-3-[4-[(1,l- S° -dimethylethoxy)-2-oxoethyl]phenyl]propanoic acid [Ac-(D,L)Phe (4-CH 2 COOtBu)] (350 mg, 1 mmol) using DCC (210 mg, 1 mmol) and HOBt (202 mg, 15 mmol) which were :S dissolved in 40 ml DMF/CH 2 C1 2 by volume and ti allowed to react for 60 min at room temperature, then washed (step 8-16) and dried to yield 1.2 g Il 25 Ac-(D,L)Phe(4-CH 2 COOtBu)-Lys-Gly-Trp-Met-Asp-N-methyl- Phe-BHA-resin. This peptidyl-resin was suspended and shaken in 50% TFA/CH2 Cl with 1% EDT (2 x 20 ml) 10 min each i time at room temperature to remove the OtBu group, washed 1' (3 x 50 ml each) with CH2Cl 2 and dried in vacuo. The peptide was cleaved from the resin by treatment with 15 ml of HF containing 1.0 ml of anisole, 0.5 ml of DTE for 2 hours at 0°C. After thorough evaporation, the resin was washed with 2 volumes of ethylacetate, then triturated with 4 x 15 ml of 30% acetic acid, filtered and lyophilized to yield 200 mg of crude peptide.

I 82 100 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C 18column. The peptide was eluted with a linear gradient (4h) of 5 to 0.022% TFA/CH 3CN of a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 12 mg of: Ac-.(D,L)Phe(4-CH COOH)-Lys-Gly-Trp-Met-Asp-N-methyl-Phe- 2_ NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino dcid analysis: Asp, 1.08(l); Gly, 1.00(l); Met, 1.00(l); Lys, 0.95(l); Phe(4-CH 2COOH), 0.92(1); N-methyl-Phe Trp, 0.70(l). Empirial Formula: C 51H 64N 1 011S M.W.

1025.18.

EXAMPLE 57 of Ac-(D,L)Phe(4-tetrazole)-Lys--GlV-Trp-Met-Asp- Q 0 0N-methyl-Phe-NH 2 1.1 g (0.33 mmol) of Fmoc-Lys-Gly-Trp-Met--Asp-N-methyl- CIO0 Phe-BRA-resin obtained from Example 1,6 was deprotected with 20% piperidine/DMF (step 1-6) using the Fmoc protocol and coupled to the compound of Example 17(c), Of*,rac.-2-acetamido-3-[4-[5-2-(1, l-dimethylethyl)-2H-tetrazoylj J*phenylipropanoic acid [Ac-(D,L)Phe(4-tetrazole-tBu)] (500 mg, 1.5 mrnol). using DCC (310 mg, 1.5 mmol), and HOBt (270 mg, 2 mmol) which were dissolved in 40 ml

DMF/CH

2 C1 2 by volume and allowed to react for 2 00 min at room temperature, then washed (step 8-16) and dried to yield 1.2 g of: AckDLPe4ttaoetu-y-l-r-e-s--ehl Phe-BHA resin which was cleaved by treatment with HF (15 ml) containing 1.0 ml anisole, 0.5 ml DTE, and 0.5 ml U~CI___LI I 83 dimethylsulfide for 2h at 0 0 C. After thorough evaporation, the resin was washed with 2 volumes of ethylacetate, then triturated with 4 x 15 ml of 30% acetic acid, filtered and lyophilized to yield 123 mg of crude peptide.

123 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30) cm micro Bondapack C18 column. The peptide was eluted with a linear gradient (4h) of 5 to 0.022% TFA/CH3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 20 mg of: Ac-(D,L)Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N-methyl-Phe- NH2' This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.00(1); Gly, 1.00(1); Met, 1.03(1); Lys, 1.01(1); Trp, o Phe(4-tetrazole); 0.97(1); N-methyl-Phe, n.d.

0 Empirical Formula: C50H62N409S. M.W. 1035.90.

D C 0 00 .EXAMPLE 58 Preparation of Ac-(D,L)Phe-(3-CH COOH)-Met-Gly-Trp- 0, 0. Met-Asp-Phe-NH o0 1.00 g (0.87 mmol) of Fmoc-Met-Gly-Trp-Met-Asp(OtBu)o 0 Phe-PAM-resin obtained from Example 42 was deprotected with piperidine/DMF (step 1-6) using the Fmoc protocol and coupled to the compound of Example 33, rac.-N-acetyl-3-[2- 30 -(1,1-dimethylethoxy)-2-oxoethyl]phenylalanine o, [Ac-(D,L)Phe(3- CH2CO2tBu)](280 mg 0.8 mmol) using DCC 0 o (165 mg. 0.8 mmol) and HOBt (200 mg. 1.5 mmol) which were dissolved in 40 ml DMF/CH 2 Cl 2 by volume and allowed to react for 60 min at room temperature. Then washed (step 8-16) and dried to yield Ac-(D,L)Phe(3-CH 2 COOtBu)-Met-Gly-Trp-Met-Asp(OtBu)- Phe-PAM resin. This peptidyl-resin was suspended and shaken I( C- I I -il- 84 in 50% TFA/CH2Cl 2 with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the OtBu groups. The peptidyl-resin was then isolated by filtration, washed (3 x ml each) with CH 2C l 2 DMF and methanol, and placed in a pressure bottle suspended in 100 ml of methanol, saturated with NH 3 at 200 and sealed. The suspension was stirred at room temperature for 3 days. After venting the excess

NH

3 the PAM-resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness to yield 456 mg of crude peptide. 80 mg of the crude peptide was purified by preparative HPLC on a (2.3x30) cm micro Bondapack C18 column. The peptide was eluted with a linear gradient (4h) of 5 to 65% 0.022%. TFA/CH3CN of a flow rate at 8 ml/min. detection at 280 nm. The main peak was collected and lyophilized to yield 14 mg of K.:o Ac-(D,L)Phe(3-CH COOH)-Met-Gly-Trp-Met-Asp-Phe- NH 0 a 2* °o a This material was homogeneous by HPLC and gave the correct o amino acid analysis and MS. Amino acid analysis: Asp 1.00 Gly 0.80 Met 2.08 Phe 1.00 Trp 0.70 Phe(3-CH 2 COOH) 1.00 Empirical Formula SC49H N 02 2' M.W. 1032.20.

49 61 9 12 2' i EXAMPLE 59 S 25 Preparation of Desamino Phe(4-CHCOOH)-Met-Gly-Trp- 2 -Met-Asp-Phe-NH 2 1.00 g (0.87 mmol) of Fmoc-Met-Gly-Trp-Met-Asp-(OtBu)- Phe-PAM resin obtained from example 42 was deprotected with 20% piperidine/DMF (Step 1-6) using the Fmoc protocol and coupled to the compound of Example 26, N-hydroxysuccinyl 3-(4-carboxymethyl)phenylpropaonate, (244 mg. 0.8 mmol) which was dissolved in 20 ml of DMF/CH 2 Cl 2 by volume and allowed to react for 24 hours at room temperature, then washed (step 8-16) and dried to yield Desamino Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp (OtBu)-Phe-PAM resin. This peptidyl-resin was suspended and shaken in l- l----au 85 TFA/CH2C12 with 1% EDT (2 x 50 ml) 10 min each time at room temperature to remove the OtBu group. The peptidyl-resin was then isolated by filtration, washed (3 x ml each) with CH2Cl DMF and methanol, and placed in a pressure bottle, suspended in 100 ml of methanol, saturated with NH 3 at -200 C and sealed. The suspension was stirred at room temperature for 3 days. After venting the excess NH 3 the PAM-resin was filtered off and washed with methanol. The combined filtrates were evaporated to dryness to yield 733 mg of crude peptide. 80 mg of the crude peptide was purified by preparation. HPLC on a (2.3 x cm) micro Bondapack C18 column. The peptide was eluted with a linear gradient (4 h) of 5 to 65% 0.022%TFA/CH3CN at a flow rate of 8 ml/min. and detection at 280 nm. The main peak was collected and lyophilized to yield 8 mg (38%) of Desamino Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH2.

This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp 1.00 Gly 1.00 Met 2.00(2); Phe 1.00 Trp 0.71 Empirical formula C 47

H

58 N80 S 2 MW 975.2.

EXAMPLE S; Preparation of Ac-(D,L)Phe(4-CH tetrazole)-Met-Gly- 25 Trp-Met-Asp-Phe-NH 2 0 002 1.5 g (0.48 mmol) Boc-Met-Gly-Trp(For)-Met-Asp(OBZ1) -Phe-BHA resin obtained from example 53 was deprotected with TFA/CH Cl 2 with 1% EDT (step 1-15) using the Boc 30 protocol and coupled to the compound of Example 17(e), •rac.-2-amino-3-[4-[[5-[2-(1,l-dimethylethyl)-2H-tetrazoyl]]methyl] phenyl]propanoic acid, [Ac-(D,L)Phe(4-CH2-tetrazole-tBu] (525 mg, 1.5 mmol). DCC (310 mg, 1.5 mmol)and HOBt (270mg, 2 mmol) which were dissolved in 50 ml DMF/CH 2 C12 by volume and allowed to react for 60 min at room temperature. At this point, the peptidyl-resin was dried under high vacuum to i 86 provide 1.7 g of Ac-(D,L)Phe(4-CH 2 tetrazole-tBu)-Met-Gly- Trp(For)Met-Asp(OBZl)-Phe-BHA-resin. 1.7 g of the resin was cleaved by treatment with 5 ml of HF containing 2.0 ml of anisole, 1.0 ml of EDT and 15 ml of dimethylsulfide for 1 hour at 0°C. After evaporation to a low volume, fresh anhydrous HF (20 ml) was distilled into the reaction vessel for a second treatment for 2 hours at 0 C. After thorough evaporation, the resin was washed with 2 volumes of ethylacetate then triturated with 4 x 15 ml of 30% acetic acid, filtered and lyophilized to yield 456 mg of crude peptide. 80 mg of the crude peptide was purified by preparative HPLC as a (2.3 x 30) cm micro Bondapack C 18 18 column. The peptide was eluted with a linear gradient (4 h) of 5 to 65%. 0.022% TFA/CH3CN of a flow rate of 8 ml/min.

3 detection of 280 nm. The main peak was collected and 0 lyophilized to yield 18 mg of Ac-(D,L)Phe(4-CH 2 tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH This material was 2 Shomogeneous by HPLC and gave the correct amino acid analysis Sand MS. Amino acid analysis Asp 1.00 Gly 1.06(1); Met 1.80(2)Phe 1.00(1); Phe(4-CH tetrazole) 0.95 Trp 0.70 Empirical Formula: C H N 0 S MW 1056.30.

49 61 13 10 2 EXAMPLE 61 25 Preparation of Ac-(D)Phe(4-CH -tetrazole)-Met-Gly-Trp- Met-Asp-Phe-NH 2 2 mg of Ac-(D,L)-Phe(4-CH -tetrazole)-Met-Gly-Trp- Met-Asp-Phe-NH 2 obtained from Example 60 were dissolved in 30 0.5 ml of 0.2N NH OH and applied to (2.3 x 30 cm) micro 0 4 Bondapak C-18 column from E.S. Industries. The column was prev.ously equilibrated with 1% CH3CN/0.01M NH4OAc and eluted with a step gradient of 1-20% CH CN/O.01M NH4 OAc over 5 min at 8 ml/min, then 20-30% CH3CN/O.01M NH4 Ac over 120 min at 8 ml/min. The 280 nm absorption of the column effluent was monitored. Two peaks were detected eluting at 51 min and 55 min. Fractions containing these 87 peaks were pooled and lyophilized to 1 mg of white powder.

Analysis by Glass Capillary Gas Chromatography shows that the compound eluting at 51 min retention time contains the (D)-Phe(CH -tetrazole) enantiomei. The purity of this compound was also confirmed by analytical HPLC. amino acid analysis, UV and IR.

EXAMPLE 62 Preparation of Ac-Phe(4-CH 2 -tetrazole)-Met-Gly-Trp-Met- Asp-Phe-NH From Example 61, the compound eluting at 55 min retention time was analyzed by Glass Capillary Gas Chromatography and shown to contain the (L)-Phe(4-CH 2 -tetrazole) enantiomer. The purity of this 4 compound was also determined by analytical HPLC, amino acid S'analysis, UV and IR.

EXAMPLE 63 £1 I 2 Trp-Nle-Asp-N-methyl-Phe-NH 2 25 1 g (0.37 mmol) Boc-N-methyl-Phe-BHA resin was suspended and shaken in TFA/CH 2 Cl 2 by volume (3 x 40 ml) min each time at room temperature to remove the Boc-group.

The product was isolated by filtration and washed (3 x 50 ml each) with CH2C1l2 8% DIEA in CH2Cl 2 and CH C1 2 to give the free base of N-methyl-Phe-BHA resin. This was subjected to sequential solid phase peptide synthesis using the DCC/HOBt procedure. At Step 16 the Boc-amino acid, DCC and HOBt were added with the corresponding reaction times as follows: Boc-Asp(OBzl)-OH (485 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml 1:1 by volume DMF/CH Cl 2 and allowed to couple for min at room temperature. Boc-Nle-OH (350 mg, 1.5 mmol), DCC L_ .i ~Li--~---rrasaua~cll-maloxr~~~r- j4x-)um~ 88 (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2C1l and allowed to couple for 60 min at room temperature. Boc-Trp(For)-OH (500 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of DMF, and allowed to couple for 60 min at room temperature. Boc-Gly-OH (270 mg, mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH 2 C1 2 and allowed to couple for 60 min at room temperature.

Boc-Nle-OH (350 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH 2 Cl 2 and allowed to couple for 60 min at room temperature. The compound of Example (17e) rac.-2-acetamido-3-[4-[[5-[2-(1,1-dimethylethyl)- 2H-methyl]-tetrazoyl]]phenyl]propanoic acid (530 mg, mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were 0 dissolved in 30 ml of 1:1 by volume DMF (CH 2 C12) and O allowed to couple for 60 min at room temperature. At this point the peptidyl-resin was dried under high vacuum to 0 provide 1.7 g of Ac-(D,L)-Phe(4-CH2-tetrazole -tBu)-Nle-Gly-Trp(For)- Nle-Asp(OBzl)-N-methyl-Phe-BHA resin. 1.7 g of the resin was cleaved by treatment with 5 ml of HF containing 2.0 ml of anisole, 1.0 ml of EDT and 15 ml of dimethylsulfide for 1 h at 0 C. After evaporation to a 25 low volume, fresh anhydrous HF (20 ml) was distilled into the reaction vessel for a second treatment for 2 h at 0°C.

After thorough evaporation, the resin was washed with 2 volumes of ethylacetate, then triturated with 4 x 15 ml of acetic acid, filtered and lyophilized to yield 530 mg of crude peptide.

100 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30 cm) micro Bondapak C-18 column. The peptide was eluted with a linear gradient (4 h) of 5 to 0.022% TFA/CH 3 CN of a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 10 mg of Ac-(D,L)-Phe(4-CH2-tetrazole)-Nle- I~ 89 Gly-Trp-Nle-Asp-N-methyl-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 0.98 Gly, 0.96 Nle, 2.00 N-methyl-Phe, Trp, Phe(4-CH 2 tetrazole), 0.90 Empirical formula C52H67N13010. M.W. 1034.12.

EXAMPLE 64 Preparation of Ac-(D)Phe(4-CH 2 -tetrazole)- Nle-Gly-Trp-Nle- Asp-N-methyl-Phe-NH 2 2 mg of Ac-(D.L)-Phe(4-CH2-tetrazole)-Nle-Gly-Trp-Nle- Asp-N-methyl-Phe-NH 2 from example 63 were dissolved in ml of 0.2N NH40H and applied to (2.3 x 30 cm) micro oBondapak C-18 column from E.S. Industries. The column was °a previously equilibrated with 1% CH 3 CN/0.01M NH4OAc and 0 0 eluted with a step gradient of 2-25% CH3CN/0.01M NH OAc 0 0 3 4 o° over 5 min at 8 ml/min, then. 25-40% CH3CN/0.01M NH4 OAc over 120 min at 8 ml/min. The 280 nm absorption of the column effluent was monitored. Two peaks were detected at 41 min and 44 min. Fractions containing these peaks were pooled and lyophilized to 1 mg of white powder. Analysis by Glass Capillary Gas Chromatography shows that the compound eluting at 41 min retention time contains the (D)Phe(4-CH2-tetrazole) enantiomer. The purity of this compound was also confirmed by analytical HPLC, amino acid analysis, UV and IR.

EXAMPLE Preparation of Ac-Phe(4-CH -tetrazole)-Nle-Gly-Trp- Nle-Asp- N-methyl-Phe-NH From Example 64, the compound eluting at 44 min retention time was analyzed by Glass Capillary Gas Chromatography and shown to contain the (L)Phe(4-CH 2 L_ I -i 90 tetrazole) enantiomer. The purity of this compound was also determined by analytical HPLC, amino acid analysis, UV and

IR.

EXAMPLE 66 Desamino-Phe(4-CH -tetrazole)-Nle-Gly-Trp-Nle-Asp-N-methyl- Phe-NH 1.2 g (0.37 mmol) Boc-Nle-Gly-Trp(For)-Nle-Asp(OBzl)- N-methyl-Phe-BHA-resin obtained from Example 63 was deprotected with TFA/CH 2 C12 by volume (Steps 1-15) using the Boc-protocol and coupled to the compound of Example 36, 4-[[2-(l,1-dimethylethyl)-2H-tetrazol -5-yl]methly] benzenepropanoic acid (500 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) which were 0° dissolved in 50 ml of DMF/CH C1 2 by volume and allowed to react for 60 min at room temperature, then washed 0 (Steps 17-27) and dried to yield 1.4 g Desamino-Phe(4-CH 2 I 20 tetrazole-tBu)-Nle-Gly-Trp(For)-Nle-Asp(OBzl)-N-methyl-Phe- BHA-resin. 1.4 g of the resin was cleaved by treatment with HF using the same condition as described in Example 63 to yield 400 mg of crude peptide. 100 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30 cm) micro I 25 Bondapak C-18 column, The peptide was eluted with a linear gradient (4 h) of 5 to 65% 0.022% TFA/CH3CN of a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 16 mg of SDesamino-Phe(4-CH 2 tetrazole)-Nle-Gly-Trp-Nle-Asp-Phe-

NH

2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.01 Gly, 1.00 Nle, 1.90 Trp, 0.80 N-methyl-Phe, n.d. Empirical formula: C50H64 N 0 M.W. 977.15.

91 EXAMPLE 67 Desamino-Phe(4-tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH 1.4 g (0.48 mmol) of Boc-Met-Gly-Trp(For)-Met-Asp(OBzl)- Phe-BHA-resin obtained from Example 53 was deprotected with TFA/CH Cl 2 by volume (Steps 1-15) using the Boc-protocol and coupled to the compound of Example 41 4-[2-(1,1-dimethlethyl)-2H-tetrazol-5-yl]-benzenepropanoic acid (475 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) which were dissolved in 50 ml of DMF/CH2Cl 2 by volume and allowed to react for min at room temperature, then washed (Steps 17-27) and dried to yield 1.52 g of Desamino-Phe(4-tetrazole-tBu)-Met- Gly-Trp(For)-Met-Asp(OBzl)-Phe-BHA-resin. 1.52 g of the resin was cleaved by treatment with HF using the same O o conditions as described in Example 63 to yield 340 mg of crude peptide. 100 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30 cm) micro Bondapak C-18 column. The peptide was eluted with a linear gradient (4 h) of 5 to 65% 0.022% TFA/CH3CN of a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 15 mg of Desamino-Phe(4tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.07 Gly, 0.97 41t Met, 2.00 Phe, 1.10 Trp, n.d. Empirical formula C 6H56 N 209S 2 M.W. 985.16.

EXAMPLE 68 Preparation of Desamino-Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp- N-methyl-Phe-NH 2 1.1 g (0.33 mmol) of Fmoc-Lys-Gly-Trp-Met-Asp-N- I methyl-Phe-BHA-resin obtained from Example 56 was deprotected with 20% piperidine/DMF (Steps 1-6) using the L t 92 Fmoc protocol and coupled to the compound of Example 41 4-[2-(l,l-dimethylethyl)-2H-tetrazol-5-yl]-benzenepropanoic acid tetrazole (475 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) which were dissolved in 50 ml of DMF/CH2C12 by volume and allowed to react for min at room temperature, then washed (Steps 8-16) and dried to yield 1.3 g of Desamino-Phe(4-tetrazole-tBu)-Lys-Gly-Trp- Met-Asp-N-methyl-Phe-BHA-resin. 1.3 g of the resin was cleaved by treatment with HF using the same conditions as described in Example 68 to yield 439 mg of crude peptide.

150 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30 cm) micro Bondapak C-18 column. The peptide was eluted with a linear gradient (4 h) of 5 to 65% 0.022% TFA/CH3CN.of a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilzed to yield mg of Desamino-Phe-(4-tetrazole)-Lys-Gly-Trp- Met-A p-N-methyl-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS.

o Amino acid analysis: Asp, 0.60 Gly, 0.92 Met, 1.00 0o o Lys, 0.60 Trp, 0.90 N-methyl-Phe, n.d.

Empirical formula C48H59N308S. M.W. 978.17.

EXAMPLE 69 Preparation of Desamino-Phe(4-CH -COOH)-Met-Gly-Trp-Met- Asp-N-methyl-Phe-NH 2 g of Fmoc-PAL-resin (substitution 0.38 mmol/g) was suspended and shaken in 20% piperidine/DMF (Steps 1-6) using the Fmoc protocol and coupled to Fmoc-N-methyl-Phe (2.4 g, 6 mmol) using DCC (1.25 g, 6 mmol) and HOBt (1.2 g, 9 mmol) which were dissolved in 100 ml DMF/CH 2 C1 2 by volume and allowed to react for 12 h at room temperature, then washed (Steps 8-16) and dried to yield 5.5 g of Fmoc-Nmethyl-Phe-PAL-resin. The substitution was determined by the Gisin method to be 0.34 mmol/g. A portion, 1 g (0.34 mmol) of the Fmoc-N-methyl-Phe-PAL-resin was subjected to 93 sequential solid phase synthesis using the Fmoc-protocol.

All couplings except the last residue were performed using DCC/HOBt procedure. At Step 7 the Fmoc-amino acids, DCC and HOBt were added with the corresponding reaction times as follows: Fmoc-Asp(OtBu)-OH (615 mg, 1.5 mmol), DCC (310 mg, mmol) and HOBt (270 mg, 2 mmol), were dissolved in 20 ml of 1:1 by volume DMF/CH2C2l and allowed to couple for min at room temperature. Fmoc-Met-OH (550 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH 2 C12 and allowed to couple for 60 min at room temperature.

Fmoc-Trp-OH (650 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for 60 min at room temperature. Fmoc-Gly-OH (450 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in oo 0 ml of 1:1 by volume DMF/CH Cl and allowed to couple for o 9 2 2 60 min at room temperature. Fmoc-Met-OH (550 mg, 1.5 mmol), SDCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were 00 09 S 20 dissolved in 20 ml of 1:1 by volume DMF/CH Cl 2 and o allowed to couple for 60 min at room temperature. At this point the Fmoc-Met-Gly-Trp-Met-Asp(OtBu)- N-methyl-Phe-PAL-resin was suspended and shaken in ooa o piperidine/DMF (Steps 1-6) using the Fmoc-protocol and 25 coupled to the compound of Example 26 N-hydroxysuccinyl 3 -(4-carboxymethyl)phenylpropanoate (610 mg, 2 mmol) which o0 was dissolved in 20 ml DMF/CH2 C 2 by volume and allowed to react for 6 h at room temperature, then washed (Steps 8-16) and dried to yield 1.40 g of 30 Desamino-Phe(4-CH 2 COOH)-Met-Gly-Trp-Met- Asp(OtBu)-N-methyl-Phe-PAL resin. This peptidyl-resin was suspended and shaken in 50 ml of TFA/EDT/CH2 C2 (14/1/5) by volume for 1 hour at room temperature, then the PAL resin was filtered off and washed with 20 ml of TFA/CH2C12 by volume. The combined filtrates were evaporated to dryness, precipitated with ether, filtered off and dried to yield 268 mg of crude peptide. 135 mg of the crude peptide i 94 Swas purified by preparative HPLC on a (2.3 x 30 cm) micro Bondapak C-18 column. The peptide was eluted with a linear gradient of 5 to 65% of 0.022% TFA/CH3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 12 mg of Desamino-Phe(4-

CH

2 COOH)-Met-Gly-Trp-Met-Asp-N-methyl-Phe-NH2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 1.00 Gly, 1.05 Met, 1.80 N-methyl-Phe, Trp, n.d.

Empirical formula: C H N0 S M.W. 989.15.

48 60 8 11 2 EXAMPLE Preparation of Desamino-Phe(4-CH COOH)-Nle-Gly-Trp-Nle- Asp-N-methyl-Phe-NH 2 1 g (0.34 mmol) of Fmoc-N-methyl-Phe-PAL-resin obtained from Example 69 was subjected to sequential solid phase synthesis using the Fmoc-protocol. All couplings except the 20 last residue were performed using the DCC/HOBt procedure.

At Step 7 the Fmoc-amino acid, DCC and HOBt were added with the corresponding reaction times as follows: Fmoc-Asp(OtBu)-OH (615 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) S. and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by 25 volume DMF/CH2C12 and allowed to couple for 60 min at room temperature. Fmoc-Nle-OH (520 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in ml of 1:1 by volume DMF/CH2C12 and allowed to couple 2 2 for 60 min at room temperature. Fmoc-Trp-OH (650 mg, mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for 60 min at room temperature.

Fmoc-Gly-OH (450 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2C1 2 and allowed to couple for 60 min at room temperature. Fmoc-Nle-OH (520 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 1.5 mmol) were dissolved in i 95 ml of 1:1 by volume of DMF/CH2Cl 2 and allowed to couple for 60 min at room temperature. At this point the Fmoc-Nle-Gly-Trp-Nle-Asp(OtBu)-N-methyl-Phe-PAL-resin was suspended and shaken in 20% piparidine/DMF (Steps 1-6) using the Fmoc protocol and coupled to the compound of Example 26 N-hydroxysuccinyl 3-(4-carboxymethyl)phenylpropaonoate (610 mg, 2 mmol) which was dissolved in 20 ml DMF/CH2C Cl2 by volume and allowed to react for 6 h at room temperature, then washed (Steps 8-16) and dried to yield 1.45 g of Desamino-Phe(4-CH2COOH)-Nle-Gly-Trp- Nle-Asp-(OtBu)-N-methyi-Phe-PAL-resin. This peptidyl-resin was suspended and shaken in 50 ml of TFA/EDT/CH2Cl 2 (14/1/5) by volume for 1 h at room temperature, then the PAL resin was, filtered off and washed with 20 ml TFA (CH2C12 by volume. The combined filtrates were evaporated to dryness, precipitated with ether, filtered off and dried to yield 240 mg of crude peptide.

S120 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30 cm) micro Bondapak C-18 column. The peptide was eluted with a linear gradient of 5 to 65% of 0.022% TFA/CH3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 10 mg of Desamino-Phe(4-CH 2 COOH)-Nle-Gly-Trp- 0.0'0. 25 Nle-Asp-N-methyl-Phe-NH 2 This material was homogeneous by HPLC and gave the correct amino acid analysis and MS.

.4 Amino acid analysis: Asp, 0.96 Gly, 0.93 Nle, 2.00 N-methyl-Phe, Empirical formula: C50 H64N 011 M.W. 953.11.

o* 0 EXAMPLE 71 Preparation of Desamino-Phe(4-CH COOH)-Nle- (D)Ala-Trp-Nle-Asp-N-methyl-Phe-NH 1.25 g (0.43 mmol) of Fmoc-Trp-Nle-Asp(OtBu)-N-methyl- Phe-PAL-resin obtained from Example 70 was subjected to -96 sequential solid phase synthesis using the Fmoc protocol.

All couplings except the last residue were performed using the DCC/HOBt procedure. At Step 7 the Fmoc-amino acid, DCC and HOBt were added with the corresponding reaction times as follows: Fmoc(D)-Ala-OH (480 mg, 1.5 mmol), DCC (310 mg, mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume DMF/CH2Cl 2 and allowed to couple for min at room temperature. Fmoc-Nle-OH (520 mg, 1.5 mmol), DCC (310 mg, 1.5 mmol) and HOBt (270 mg, 2 mmol) were dissolved in 20 ml of 1:1 by volume of DMF/CH Cl 2 and allowed to couple for 60 min at room temperature. At this point the Fmoc-Nle-(D)Ala-Trp-Nle-Asp(OtBu) -N-methyl-Phe-PAL-resin was suspended and shaken in piperidine/DMF (Steps 1-6) using the Fmoc protocol and coupled to the compound of Example 26 N-hydroxysuccinyl 0 3-(4-carboxymethyl)phenylpropanoate (610 mg, 2 mmol) which was dissolved in 20 ml DMF/CHC1 2 by volume and a 2 2 :0 °o allowed to react for 6 h at room temperature, then washed S°(Steps 6-16) and dried to yield 1.5 g of 0 Desamino-Phe(4-CH 2 COOH)-Nle-(D)-Ala-Trp-Nle-Asp(OtBu)- N-methyl-Phe-PAL-resin. This peptidyl-resin was suspended and shaken in 50 ml of TFA/EDT/CH Cl (14/1/5) by volume for 1h at room temperature, then the resin was filtered off and washed with 20 ml TFA/CH2Cl by volume. The combined filtrates were evaporated to dryness, precipitated with ether, filtered off and dried to yield 300 mg of crude peptide.

150 mg of the crude peptide was purified by preparative HPLC on a (2.3 x 30 cm) micro Bondapak C-18 column. The peptide was eluted with a linear gradient of 5 to 65% of 0.022% TFA/CH3CN at a flow rate of 8 ml/min, detection at 280 nm. The main peak was collected and lyophilized to yield 20 mg of Desamino-Phe(4-CH COOH)-Nle-(D)Ala- Trp-Nle-Asp-N-methyl-Phe-NH This material was homogeneous by HPLC and gave the correct amino acid analysis and MS. Amino acid analysis: Asp, 0.95 Ala, 1.03 L i 97 Nle, 2.02 N-methyl-Phe, Trp, Empirical formula: C 5 1

H

6 6

N

8 01; M.W. 967.13.

EXAMPLE 72 In Vitro Receptor Binding Assay Frozen bovine striatum (approx. 5 g) or fresh rat pancreas (approx. 5 g) cleaned of fat and extraneous tissue were homogenized in HEPES buffer #1 (10 mM HEPES 130 mM NaC1 5 mM MgCl 2 pH 7.4) using 35 parts buffer per 1 part tissue on a wet weight/volume basis (approx. 175 ml).

The tissue was homogenized 2 x for approx. 15 sec. at 0 0

C

using a Polytron homogenizer at a setting of 6. The tissue was isolated by centrifugation at 48,000 x g for 10 min at OOC. The resulting tissue pellet was resuspended in HEPES buffer #2 (10 mM HEPES 130 mM NaC1 5 mM MgCl 2 1 mg/L o' phenylmethanesulfonyl fluoride (PMSF) 200 mg/L Bacitracin): 1 part striatal tissue (original wet weight) per 80 parts buffer and 1 part pancreas tissue (original wet weight) per 500 to 1000 parts buffer.

Incubation was initiated by combining various concentrations of native CCK-8 or peptides of the invention 3 25 with H-CCK-8-(SO 3 H) (final cone. 0.15 nM) and tissue homogenate (striatum approximately 0.26 mg protein in 2 ml final volume; pancreas approximately 0.100 mg protein in 1 ml final volume). Samples were incubated for 30 min at 0 C and the incubation terminated by pouring the mixture onto a pre-wetted Whatman GF/B filter on a Sandbeck Vacuum Filtration Manifold. The incubation tubes were washed with 2 x 3 ml of ice-cold HEPES Buffer #2 and the wash filtered through the GF/B filter. The filter was air dried for 10 min and then placed in a scintillation vial with 12 ml of DuPont/NEN Aquasol scintillation cocktail. The vials were shaken overnight and then counted using a liquid scintillation spectrometer. Non-specific binding was 98 determined in the presence of 1 micromolar native CCK-8 and subtracted from all samples to determine specific binding.

The concentration of peptide necessary to inhibit 50% of 3 total specific H-CCK-8-(SO3H) binding (IC 50 value) was determined by log-probit analysis. The results are summarized in Table I.

EXAMPLE 73 Two-Meal Feeding Assay Male Sprague-Dawley (CD) rats weighting 180-200 grams (Charles River Breeding Laboratories) were acclimated to a 12h light/dark cycle (6 a.m. to 6 in a room kept at 22 0 C. They were subsequently fasted for two days, weighed, oo. placed in individual cages, and a four-day period of meal Straining was begun. During this time the rats were given ground laboratory chow (Purina Lab Chow) in jars for one S. hour from 9:00 a.m. until 10:00 the jars were removed S 20 from 10:00 a.m. to 12:00 and placed back in the cages from 12:00 until 1:00 p.m. Under this meal feeding regime, most rats learn to eat approximately as much per day during the two hours they have access to food as rats which have food ad libitum over the entire 24-hour day. On the Ij 25 fourth day, the rats were weighted again, and any which lost more than five grams body weight were excluded from the test. The animals were then distributed into experimental (n 5 to 6) and control groups (n 6-12), but not matched for body weight.

Peptides of the invention were suspended either in saline, if soluble, or in 0.5% DMSO/saline, if insoluble, at concentrations of 0 to 320 pg/ml/kg body weight and were administered intraperitoneally 15 min before the first meal on day 5 of meal feeding. The rats were then given their meals as they had been during the previous four days, and the food cups were weighed both before and after each meal p~1(L~ ii i 99 to determine food consumption. Food intake was expressed as a mean and standard error of the mean in percent of control values for the various groups. The treated groups were compared to the control groups by t-test analysis. The results are summarized in Table 1.

While the invention has been described in connection with the preferred embodiment, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the appended claims.

o 0o o o 1Y1EI(PIT 1) Fqtie Ex. t. Bovine Striatun (ni) Iabt FW-creas (ri) Flxzd Intake tliro--- ~g Of amtiirl (D,L )Fhe( 3 ~i-tY(y-q~Pt-s-Eie2 GJ(-8 1-3.2 1-4.6 42 32 128 27+17*** 149+6*** 1259-12 98+14 43 34 62 320 1060 43+11*** 98+9 11642 118+13 inj~i 12349 146T6* APc-( D)R-je(4-0JJZI1) Vt-TPn-t~~p -A:4H 2 44 68 210 89+6 6A-4 62 30 79+12 14046* ff+6**k 1f6** 46 47 3.6 97+15 95A4 5345*** 96-f6 157+18** j33T[jJ* 14246* 1800-10 17216** 141+12* 47 145 (pege 4 4 4 Striatun WMi) Reptib Panreras (rim) Dose~ microrau'kq Food] Intake 1st. Mal 2M Mal1 of Ouitrol (crc 2 Fe(4CgJH R e t-Mn- -APff- 48 54 2.6 49 10000 1000 10000 _3600 76+6* 93+-3 714-8** 97+3 66i* 16+6*** 1394-1b 17IT20* 116+14 984-15 1654-14** 228fW16~** 10:F75 1-1248 1694T22** Pc- (D,L)Fhe( 4-CT21)-4t-'1p-A-t-Ap-He4- 2 620 390 66 81 103f5 1141-7 54+I6*** 186+3*** 201+3*

L~.

04 ThWIE I (peqj 3) Pqtid E. b. Buvine Striabtun (rim) Ibat P3ares (mt) Doxc~rr}e Fbod Irtake of Qnrtol Ac- e(4-tetrazole) t~-M.tp-rt-Agp-R-41 2 Pc--(D)Hi)e( 4-tetrazoe)--~-'f Trp-F-t-A---rE+ry1- HeitI I Pc-(D,L)Fhe(3-GI 2 CY)-+--t-Gy-'ft+t-Aq-The-U 2 53 1.6 54 507 120 0.51 3.0 56 10000 4650 49+11** 289*** 86+16 70410 32+10*** 89+7 83** 75f6** 144+8** 146412** 131+8* 140+25 145+12 156+8k 19f10* 98+9 12f+2 1601-14* 148* 580 1100 53 23 22 57 169f+11w dib*i* 16j313 59+5** lrik*** 197+21** 12+14 0 @0

N)

01 000 000 @00 0 C a 0 0 2 0 ~0 OP 02020 2 0 2 00

N)

0 RFptie rnUE I (paTj 4) StriatT (mt) Pat~e (ni)ea Mirosre k Flxd Intake 1st ma1 2d mm1 Of Gchtro1 Pt--(DL)Rie(4-,CH-r-tetrazole)-Vkat--Qyjftp-ret-Asp- R)-4U2 60 340 63f9** 12gt*** 14%115 16519 pa a p Q~p aoo p a a mNE I (page Striatim (rnM) Pat Pancreas (mi) Rxod Intake Micrograiykg Of ctritrol AcR )ie4a-ettaz411 2 -M -ir 4t--p AN-rey-~thetrzl)-M1-1 2 Tpie-s N-frethyl-Rhe-MT 2 61 62 380 62 18 67 80+6* 47F+3*** 85+6* 254A*** 53+-10** 19T5*** 112+10 56T6**-* 21f6* 119+28 15949q* 17+21* 118+9 12S99*** 17Q+12** 179+4* 171+3~* 141+17 102+9 146412* 17345*** 20-F+20*** 63 260 64 160

CA)

01i o 0 o oo.

000 00. UMa I (page. 6) Striatin (rim) Fbt Pancreas (ni) Doie Mictxgrm}'g Food Intake 1St Mal 71d Mal of CQntrol Las ardrn fie( 4-CH 2 -tetrazole)-Neayf-N>4-Ap- Eeas anim Pne( 4-tetrooe)-Lys-GMy-'fp-t4Et--As- N-M±Iry-Rh-4H 2

II

69 260 66 160 8611 94+19 66* 133+1-1* 1091-16 17SR~6** I-i+14** 67 5.1 35 68 1400 2900 99+9 116+6 24T9*** 77*8* 24T3* 165+18** 17i+14* gryg d 2T~o- d 1T00*0 d s aorqun @M afi ciap uap qwni@gtp 41pmjiifis s 3n *9+601 69+ET1 *E1+LS1i 01+LZT 8+901 V+S1 6ff 6 0081 0014T TL #49+BL 6+66 1+66 ~fN-~~i-TALP~,&N OfEC oW 'onuom T)UUIEx 40 6 pfou leaN 131 a~c (:Iq @Mice 'M *>G (L GEai) 1 2 dd 0 C\j 0 0 00 0 o 0 C C 000 .00 0 0 0 0 0 0 000 000 00- 0 0 00 0 00 0 0

Claims (12)

1. An analog of a tyrosine sulfate- or tyrosine phosphate residue(s) containing peptide wherein one or more of such residue(s) is/are replaced by residue(s) of the formula A Y z Z wherein one of A or B is selected from the group consisting of: 2 s-CO 2 R 1 or, N -[C(Ro) 2 r< N N N R 2 and the other of A or B is H; Z is H, -NH 2 lower alkyl, -NHCOR 3 -NHCO2 R4 or 300 O R 9 0 IL i. la~nariarra*a~ -~--xearr~l iii i 108 R 0 is R 1 is R 2 is H or F; H, substituted or unsubstituted lower alkyl with the substituents selected from the group consisting of hydroxyl, halogen, or aryl; H, lower alkenyl, lower alkyl or lower alkyl substituted by 1 to 3 aryl groups; H, lower alkyl, alkyl substituted by one or two aryl groups or aryl; lower alkyl or alkyl substituted by one or two aryl groups; each independently H, lower alkyl, or taken together and including the carbon atoms to which they are attached may form a six numbered ring which may be aromatic; R 3 is R 4 is 0444 a a 6 fi a I 4 i I and are and r are 0, 1 or 2; I and Y is -COOH, alkoxycarbonyl, -CONH 2 or -CO-; with the proviso that if Z is other than -NH- then Y is or if Y is other than -CO- then Z is and pharmaceutically acceptable salts thereof. c i r i 1 i ,i i ii 109

2. A compound as claimed in claim 1 which is an analog of a tyrosine sulfate residue(s) containing peptide.

3. A compound as claimed in claim 2 which is an analog of cholecystokinin (CCK).

4. The compound of claim 3 wherein the tyrosine sulfate residue(s) of CCK is/are replaced by a residue of Formula I wherein Z is H, NHCOR 3 or NHCO2R 4 and R 3 10 is lower alkyl, R 4 is lower alkyl optionally substited with one to three aryl groups. The compound of claim 4 wherein R is CH 3

6. The compound of claim 5 wherein one of A or B is -[C(Ro)2]s-CO 2 R 1 and the other of A or B is hydrogen.

7. The analog of claim 6 wherein A is 2 ]s-CO 2 R 1 and B is H.

8. The compound of claim 7 wherein s is O and R, is i 64 6 6 6

9. The compound of claim 8 having the formula: Ac-(D,L)Phe(4-COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 The compound of claim 8 having the formula: Ac-(D)Phe(4-COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2

11. The compound of claim 8 having the formula: Ac-Phe(4-COOH)-Met-Gly-Trp-Met-Asp-Phe-NH e 110

12. The compound of claim 7 wherein s is 1.

13. The compound of claim 12 wherein R 0is H or F. 14 Th comoun ofclai 13wheein 1 s H

514. The compound of claim 13 wherein R 0is H. 16. The compound of claim 14 herin Rh isrmuH. 16. The compound of claim 15 having the formula: Ac-(D)Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-2 17. The compound of claim 15 having the formula: 20Ac-(Phe(4-CH COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 18. The compound of claim 15 having the formula: 20Ac-p(4-C)he4COH)-Met-y-Gly-Trp-Mets-Aph-N 19. The compound of claim 15 having the formula: DeAc-n (.LPhe (4-CH 2 COOH)-y--Gly-Trp--Met-Asp-- Pmeh-PHe- The compound of claim 15 having the formula: Desamino Phe(4-CH 2COOH)-e-Gly-Trp-NMe-Asp-- melPhe-NH 2 ill 0 0 0 00 0 0 0 0 0 0 *0 0 01 22. The compound of claim 15 having the formula: Desamino Phe(4-CH 2 COOH)-Nle-(D)Ala-TrpNle-Asp-N methyl-Phe-NH 2 23. The compound of claim 13 wherein R 0is F. 24. The compound of claim 23 having the formula: Ac- L)Phe (4-CF 2 COOH) -Met--GIv-Trp-Met-Asp-Phe- NH 2 The compound of claim 13 wherein R 0is H. 26. The compound of claim 25 wherein R 1is CH 2CH 3 (COO 2 3 he N 27. The compound of claim 26 having the formula: Ac-(Phe(4-CH COOC H )-Met-Gly-Trp-Met-Asp (COOC 2H 5)-Phe--NH 2 32. The compound of claim 26 having the formula: AcPh(4CH2 2 COH 5 )-Met-Gly-Trp-Met-Asp-Pe liz 33. The compound of claim 6 wherein B is 0 2 1s-CO 2 R 1 and A is H. 34. 36. The compound The compound The compound Ac-(DL)Phe( The compound The compound The compound of claim of claim of claim 3 -COOH) -Me of claim of claim of claim 33 wherein s is 0. 34 wherein R1is H. 35 having the formula: t-Gly-Trp--Met-Asp-Phe-NH 2 33 wherein S is 1. 37 wherein R0is H. 38 wherein R Iis H. 37. 38 39 0 4 The compound of claim 39 having the formula: Ac-(DL)Phe(3-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phie- NH2 41. The compound of claim 5 wherein one of A or B is N, -{C(RO) 2 Ir~ N N and the other of A or B is hydrogen. 113 42. The compound of claim 41 wherein A is N N and B i s H. 43. The compouad of claim 42 wherein r is 0. 44. The compound of claim 43 wherein R 2is H. The compound of claim 44 having the formula: Ac-(DL)Phe(4--tetrazole)-ZMet-Gly-Trp-Met-Asp-Phe- NH 2 46. The compound of claim 44 having the formula: Ac-(D)Phe(4-tetrazole)-Met-Gly-Trp--Met-Asp-Phe-NH 2 47. The compound of claim 44 having the formula: Ac-Phe (4-tetrazole )-Met-Gly-Trp--Met--Asp-Phe-NH 2 48. The compound of claim 44 having the formula: Ac-(D,L)Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N- methyl-Phe-NH2 49. The compound of claim 44 having the formula: Desamino Pie (4-tetrazo le) -Met-Gly-Trp-Met-Asp-Phe- NH 2 114 The compound of claim 44 having the formula: Desamino Phe(4-tetrazole)-Lys-Gly-Trp-Met-Asp-N- methyl-Phe-NH 2 51. The compound of claim 42 wherein r is 1. 52. The compound of claim 51 wherein R 0is H. 53. The compound of claim 52 wherein R2is H. 54. The compound of claim 53 having the formula: Ac- L)Phe(4-CH 2-tetrazole) -Met-Gly-Trp-Met-Asp -Phe-NH 2 The compound of claim 53 having the formula: Ac-(D)Phte-(4-CH 2-tetrazole)-Met-Gly-Trp-Met-Asp- Phe-NH 2 56. The compound of claim 53 having the formula: Ac-Phe- (4-CH 2- tetrazo le) -Met-Gly-Trp-Met-Asp- Phe-NH 2 57. The compound of claim 53 having the formula: Ac-(DL)Phe-(4-CI 2-tetrazole) -Nle-Gly-Trp-Nle-Asp- N-methyl-Phe-NH 2 58. The compound of claim 53 having the formula: Ac- (D)Phe(4-CH 2-tetrazole)-Nle-Gly-Trp-Nle-Asp- N-methyl-Phe-NH 2 115- 59. The compound of claim 53 having the formula: Ac-Phe(4-CH2-tetrazole)-Nle-Gly-Trp-N1e-Asp-N-methyl-Phe-NH 2 The compound of claim 53 having the formula: Desamino-Phe(4-CH2-tetrazole)-Nle-Gly-Trp-N1e-Asp-N-methyl-Phe-NH. 61. The compound of claim 53 having the formula: Desamino-Phe(4-CH2-tetrazole)-N1e-(D)Ala-Trp-Nle-Asp-N-methy1-Phe-NH 2 62. A compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide as defined in claim 1, said compound having the formula: B' A' Z' O-R' 0 wherein one of A' or, is selected from -[C(Ro)2 s-CO 2R', or NN 2 1'm 0 0 N N -JC(Ro) 2 Jr R 2 and the other of A' or B' is H wherein R 0 is H or F, R 1 is H, substituted or unsubstituted lower alkyl with the substituents selected from hydroxyl, halogen, or aryl, R 2 is H, lower alkenyl, lower alkyl or lower alkyl substituted by 1 to 3 aryl groups, r is 0-2 s is 1-2 R' is H, lower alkyl or lower alkyl substituted by 1 to 3 aryl groups, Z' is H, lower alkyl, -NH 2 -NHCOR 3 -NHCO 2 R 4 or O R9 o y"0 LMM/597Z ~L 116 and R3 and R4 are H, lower alkyl or lower alkyl substituted by one, two or three aryl groups; and R 5 and R 9 are each independently H, lower alkyl, or taken together and including the carbon atoms to which they are attached may form a six membered ring which may be aromatic. 63. A compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide as defined in claim 1, said compound having the formula: 3 Z OH wherein Z' and R 1 are as defined in claim 62. 64. A compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide as defined in claim 1, said compound having the formula: I CO.H :.co .0 3 Z'I O-R' I: "O wherein R' and Z' are as defined in claim 62. 65. The compound of claim 62 wherein one of A' or B' is N S-[C(Ro)23r R2 and the other of A' or B' is H. 66. A compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine pho:phate residue(s) containing peptide as defined in claim 1, said compound having the formula: LMM/597Z 117 A' 2' wherein and R' are as defined in claim 62 and R 8 is H or lower alkyl. LMM/597Z 11IL8 67. The compound of claim 66 wherein A' is -[C(RoD) 2 1r N and B'I is H, 62. and Ro and R 2are as defined in claim 68. The compound of claim 67 wherein r=0. G9. The R 8 is H said compound of claim 68 wherein R' is t-butyl and compound having the formula: 0 0 0) 00 0 C(CH 3 3 N-N 11 N C0 2 C(CH 3 3 119- A compound when used for the preparation of an analog of a tyrosine sulphate- or tyrosine phosphate residue(s) containing peptide as defined in claim 1, said compound having the formula: A' R8 S 5 wherein and R' are as defined in the second embodiment and R 8 .Q is H or lower alkyl. 71. The compound of claim 70 wherein A' is 6 aN -C(RF,) 2 ]r N R2 and B' is H and R O r, and R are as defined in claim 62. *o 10 72. The compound of claim 71 wherein r=0. 73. The compound of claim 72 wherein R' is lower alkyl and R 2 is «t-butyl said compound having the formula: ,C(CHa) 3 N-N II CO 2 R' LMM/597Z c ;i 120 74. The compound of claim 70 wherein R' is H and R 2 is t-butyl said compound having the formula: ,C(CH 3 3 N-N In N /N Cc 2 H The compound of claim 71 wherein r=l and R 0 is H. 0 0 0 0 0 0 0 o 76. The compound of claim 75 wherein R' is lower alkyl and R is t-butyl said compound having the formula: 00 2 So a 0 4 00 t NzN N-C(CH3) 3 0 3 O-R' 121 77. The compound of claim 76 wherein R' is H and R 2 is t-butyl said compound having the formula: N N-C(CH 3 3 N S'ix" C0 2 H 78. A process for the manufacture of a compound according to any S 5 one of claims 1 to 61, which process comprises treating the resin bound peptide with appropriate cleavage reagent(s) and if desired, converting such a peptide obtained into a pharmaceutically acceptable salt. 79. A pharmaceutical composition containing a compound according 4o to any one of claims 1 to 61 and a non-toxic, inert, therapeutically acceptable carrier material. S0.0 80. A pharmaceutical composition for suppressing appetite containing a compound according to any one of claims 1 to 61 and a therapeutically inert carrier material. 81. The use of a compound according to any one of claims 1 to 61 as a therapeutically active substance. 82. The use of a compound according to any one of claims 1 to 61 in the suppression of appetite. 83. Compounds as claimed in any one of claims 1 to 61 whenever prepared according to a process of claim 78. 84. A method for suppressing appetite in subjects by administering an appetite suppressing effective amount of an analog of CCK wherein the tyrosine sulfate residue is replaced by a residue of Formula I as defined in claim 1. The method of claim 84 wherein the CCK analog is a compound selected from: Ac-(D,L)Phe(3-COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D,L)Phe(4-COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 LMM/597Z 122 Ac-(D) Phe(4-COOH )-Met-G1 y-Trp-Met-Asp-Phe-NH 2 Ac-Phe(4-COOH )-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D, L)Phe(4-CH 2 COOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D)Phe(4-CH 2 COH)--Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-Phe(4-CH 2 GOOH)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-( D) Phe(4-CH 2 COOC 2 H 5 )-Met-G1 y-Trp-Met-Asp(C0C 2 H 5 )-Phe-NH 2 Ac-Phe (4-CH 2 COOC 2 H 5 )-Met-Gly-Trp-Met-Asp(C00 2 H 5 )-Phe-NH 2 Ac-Phe (4-GH 2 CH 2 000H)-Met-G1 y-Trp-Met-Asp-Phe-NH 2 Ac-(D,L)Phe(4-CF 2 000H)-Met-Gly-Trp-Met-Asp-Phe-NH 2 Ac-(D, L)Phe(4-tetrazol e)-Met-Gly-Trp--Met-Asp-Phe-NH 2 Ac-(D)Phe(4-tetrazol e)-Met-Gly-Trp-Met--Asp-Phe-NH 2 Ac-Phe(4-tet'.razol e)-Met-Gly-Trp--Met-Asp-Phe-NH 2 Desamino Phe(4-CH 000H)-Met-Gly--Trp-Met-Asp-Phe-NH 2 Ac-(D,L)Phe(3-CH 000H)-Met-G1.\-Trp-Met-Asp-Phe-NH 2 Ac-(D,L)Phe(4-CH 2 tetrazole)- 'iet-Gly-Trp-Met-Asp-Phe-NH 2 0 Ac-(D,L)Phe(4-tetraOl)-Lys-Gly-Trp-Met-Asp-N-methyl-Phe-NH 2 DeainoPhe(4-etrazol)-NLes-Gly-Trp-Met-Asp-N-methyl-Phe-NH Desamino Phe(4-CH 2 COOH)-Nle-(D)l-Trp-Nle-Asp-N-methyl-Phe-NH 2 Desamino Phe(4-tetraOl)-l-ys-ly-Trp-e-Asp-N-methyl-Phe-NH :002 2c()h(-H-erzl)Me-l-r-e-s-h- Dac- oPhe(4-tetrazole)-Met-Gly-Trp-Met-Asp-Phe-NH Deac-(DLPhe(4-tetrazole)-NL-Gly-Trp-le-Asp-N-methyl-Phe-NH 2 25Ac-(DLPhe(4-CH 2tetrazole)-Nle-Gly-Trp-Nle-Asp-N-methyl-Phe-N Desaminro-Phe(4-CH 2 -tetrazo1 e)-N1 e-Gly-Trp-N1 e-Asp-N-methyl -Plhe-NII 2 86. The method of claim 84 or claim 85 wherein the analog is administered intranasally. 87. The method of claim 84 or claim 85 wherein the analog is admini ftered parenterally. 88. An analog of CCK wherein the tyrosine sulfate residue is replaced by a residue of Formula I as defined in claim 1, which analog is substantially as hereinbefore described with reference to Example 69. LMM/5977Z i i Lt.L-C-. i- 123 89. A process for the manufacture of an analog of CCK wherein the tyrosine sulfate residue is replaced by a residue of Formula I as defined in claim 1, which process is substantially as hereinbefore described with reference to any one of Examples 42 to 71. 90. The CCK analogue product when produced by the process of claim 89. 91. A pharmaceutical composition for appetite suppression in a subject, which composition comprises an amount effective as an appetite suppressant in said subject of the CCK analog product of claim together with a pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient. 92. A method of appetite suppression in a subject, which method comprises administering to a subject in need of appetite suppression, an amount effective as an appetite suppressant in said subject of the CCK analog product of claim 90, or the composition of claim 91. o o- o DATED this TNELFTH day of AUGUST 1991 S" F. Hoffmann-La Roche Co. Aktiengesellschaft S. Patent Attorneys for the Applicant SPRUSON FERGUSON J LMM/597Z i -1